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Journal: Biology of reproduction

208

Somatic cell nuclear transfer (SCNT) is a unique technology that produces cloned animals from single cells. It is desirable from a practical viewpoint that donor cells can be collected noninvasively and used readily for nuclear transfer. The present study was undertaken to determine whether peripheral blood cells freshly collected from living mice could be used for SCNT. We collected a drop of peripheral blood (15-45 µl) from the tail of a donor. A nucleated cell (leukocyte) suspension was prepared by lysing the red blood cells. Following SCNT using randomly selected leukocyte nuclei, cloned offspring were born at a 2.8% birth rate. Fluorescence-activated cell sorting revealed that granulocytes/monocytes and lymphocytes could be roughly distinguished by their sizes, the former being significantly larger. We then cloned putative granulocytes/monocytes and lymphocytes separately, and obtained 2.1% and 1.7% birth rates, respectively (P > 0.05). Because the use of lymphocyte nuclei inevitably results in the birth of offspring with DNA rearrangements, we applied granulocyte/monocyte cloning to two genetically modified strains and two recombinant inbred strains. Normal-looking offspring were obtained from all four strains tested. The present study clearly indicated that genetic copies of mice could be produced using a drop of peripheral blood from living donors. This strategy will be applied to the rescue of infertile founder animals or a “last-of-line” animal possessing invaluable genetic resources.

Concepts: DNA, Gene, Genetics, Cell nucleus, Blood, Red blood cell, Cloning, Somatic cell nuclear transfer

26

In the Syrian hamster (Mesocricetus auratus), an animal that displays testicular regression due to short photoperiod, germ cells are removed by apoptosis during these process and the apoptotic remains are phagocytized by Sertoli cells. The aim of this work was to investigate morphologically whether the testicular regression process due to short photoperiod leads to the apoptosis of Sertoli cells and whether during testicular regression, the elongated spermatids are eliminated through phagocytosis by Sertoli cells. For this, we studied testis sections during testicular regression in Syrian hamster subjected to short photoperiod by means of using several morphological techniques using conventional light microscopy (H&E, semi-thin sections vimentin immunohistochemistry, SBA lectin, and TUNEL staining), fluorescence microscopy and transmission electron microscopy (TEM). H&E and semi-thin sections identified Sertoli cells with a degenerated morphology. Greater portion of Sertoli cells that were positive for TUNEL staining were observed especially during the mild regression (MR) and strong regression (SR) phases. In addition, TEM identified the characteristic apoptotic changes in the nucleus and cytoplasm of Sertoli cells. Moreover, during testicular regression and using light microscopy, some elongated spermatids were seen in basal position next to the Sertoli cell nucleus. This Sertoli phagocytic activity was higher in MR and SR phases. TEM confirmed this to be the result of the phagocytic activity of Sertoli cells. In conclusion, during testicular regression in Syrian hamster due to short photoperiod, when germ cells are known to be lost through apoptosis, there is morphological evidences that Sertoli cells are also lost through apoptosis, while some elongated spermatids are phagocytized and eliminated by the Sertoli cells.

Concepts: Protein, Gene, Phagocytosis, Microscope, Testicle, Transmission electron microscopy, Sertoli cell, Blood-testis barrier

24

The mammary gland undergoes development and regression over the course of the ovarian cycle under the regulation of ovarian hormones. Macrophages are implicated as local mediators of this tissue remodelling and may also affect immune surveillance and tumour incidence. To investigate cycle-related changes in macrophage phenotype, mammary gland cells from naturally cycling Cfms-Gfp mice recovered at estrus, metestrus, diestrus and proestrus were analysed by flow cytometry. Macrophage expression of MHCII was highest in the proestrus phase, with a 1.6-fold increase compared to the metestrus phase. Similarly, macrophage expression of CD204 was 1.9-fold higher at proestrus compared to estrus. Conversely, macrophage expression of NKG2D was increased at metestrus and diestrus by 7-fold and 5-fold respectively compared to estrus. To investigate hormonal regulation of macrophage phenotype, an ovariectomy and hormone replacement model was utilised. Ovariectomized mice were stimulated with exogenous estradiol and progesterone to induce early alveolar development, then given progesterone receptor antagonist RU486 to elicit alveolar bud regression. Progesterone and estradiol in combination reduced macrophage expression of MHCII and CD204 by 5-fold and 3-fold respectively, and increased macrophage expression of NKG2D by 4-fold. Administration of RU486, following estradiol and progesterone, reversed the macrophage phenotype. These results reveal an essential requirement for ovarian hormones in regulating macrophage phenotype in the mammary gland, and indicate that progesterone is particularly critical for controlling macrophage antigen presentation and immune surveillance capacity.

Concepts: Immune system, Hormone, Estrogen, Receptor, Menstrual cycle, Endometrium, Corpus luteum, Progesterone

19

ESP1/SPESP1 is a testis specific, post-meiotic gene expressed in round spermatids that encodes equatorial segment protein 1, an intra-acrosomal protein found in the acrosomal matrix and on the luminal surface of the inner and outer acrosomal membranes within the equatorial segment domain of mature spermatozoa. A comparison of testicular protein extracts with caput, corpus and caudal epididymal sperm proteins revealed striking differences in the apparent masses of SPESP1 isoforms. The predominant isoforms of SPESP1 in the testis were 77 and 67 kDa, with 47 kDa forms present to a minor degree. In contrast, SPESP1 isoforms of 47 and 43 kDa were found in caput, corpus and caudal sperm, indicating that SPESP1 undergoes noticeable mass changes during spermiogenesis and/or subsequent transport to the epididymis. On two-dimensional (2D) SDS-PAGE testicular SPESP1 isoforms resolved as a train of pIs from 4.9-5.2. Immunoprecipitated 77 kDa SPESP1 from testis reacted with the glycoprofile stain after 1 and 2-D gel electrophoresis indicating that the 77 kDa testicular isoform was highly glycosylated. One charge variant of the 67 kDa isoform was also glycoprofile positive after 2-D gel resolution. The 47 and 43 kDa isoforms of SPESP1 from epididymal sperm did not stain with glycoprofile suggesting an absence of, or few, glycoprofile sensitive glycoconjugates in epididymal SPESP1. Treatment of testicular extracts with a variety of glycosidases resulted in mass shifts in immunoreactive SPESP1 indicating that testicular SPESP1 was glycosylated and that terminal sialic acid, N- and O- glycans were present. A mixture of deglycosidase enzymes (including PNGase-F, neuraminidase, beta1-4 galactosidase, Eneo-alpha-N-acetylgalactosaminidase, and beta N-acetyl-glucosaminidase) completely eliminated the 77 and 67 kDa SPESP1 bands and resulted in the appearance of 75, 60, 55, 50, 47 and 43 kDa forms, confirming that both the 77 and 67 kDa testicular forms of SPESP1 contain complex carbohydrate residues. Treatment of caudal epididymal sperm with PNGase-F enzymes showed a faint deglycosylated band at 30 kDa, but neuraminidase did not result in any molecular shift, indicating that epididymal sperm SPESP1 did not contain sialic acid/N-acetylglucosamine residues. These findings are consistent with the hypothesis that SPSPESP1 undergoes significant glycosylation in the testis and the majority of these glycoconjugates are removed by the time sperm reach the caput epididymis. Studies of the fate of SPESP1 after the acrosome reaction localized SPESP1 to the equatorial segment region in both non-capacitated and in capacitated, acrosome reacted sperm. During capacitation, SPESP1 underwent proteolysis resulting in a 27 kDa fragment. Zona free oocytes incubated with recSPESP1 protein showed complementary binding sites on the microvillar oolemmal domain. RecSPESP1 and anti-recSPESP1 antibody inhibited in vitro fertilization.

Concepts: Protein, Molecular biology, Glucose, Sperm, Gel electrophoresis, Testicle, Reproductive system, Epididymis

8

Reduced oocyte quality has been associated with poor fertility of high performance dairy cows during peak lactation, due to negative energy balance. We examined the role of non-esterified fatty acids (NEFA), known to accumulate within follicular fluid during under- and over-nutrition scenarios, in causing endoplasmic reticulum (ER) stress of in vitro maturated (IVM) cattle cumulus-oocyte complexes (COCs). NEFA concentrations were: palmitic acid (150 μM), oleic acid (200 μM) and steric acid (75 μM). Abattoir-derived COCs were randomly matured for 24h in the presence of NEFAs and/or an ER stress inhibitor, salubrinal. Total and hatched blastocyst yields were negatively impacted by NEFA treatment compared with controls, but this was reversed by salubrinal. ER stress markers, activating transcription factor 4 (Atf4) and heat shock protein 5 (Hspa5), but not Atf6, were significantly up-regulated by NEFA treatment within whole COCs, but reversed by co-incubation with salubrinal. Likewise, glucose uptake and lactate production, measured in spent medium samples, showed a similar pattern, suggesting that cumulus cell metabolism is sensitive to NEFAs via an ER-stress mediated process. In contrast, while mitochondrial DNA copy number was recovered in NEFA treated oocytes, oocyte autofluorescence of the respiratory chain co-factor, FAD, was lower following NEFA treatment of COCs and this was not reversed by salubrinal, suggesting the negative impact was via reduced mitochondrial function. These results reveal the significance of NEFA-induced ER stress on bovine COC developmental competence, revealing a potential therapeutic target for improving oocyte quality during peak lactation.

Concepts: Protein, Metabolism, Adenosine triphosphate, Fatty acid, Mitochondrion, Cellular respiration, Cattle, Oleic acid

7

C-type natriuretic peptide (CNP) and its receptor Natriuretic peptide receptor 2 (NPR2) play a paramount role in the maintenance of oocyte meiotic arrest in antral follicles via the regulation of the intra-oocyte levels of cyclic guanosine monophosphate and cyclic adenosine monophosphate. We investigated the potential of CNP 1) to maintain oocyte meiotic arrest during a prolonged prematuration culture, and 2) to sustain acquisition of developmental competence of immature cumulus-oocyte complexes (COCs). Compact COCs were collected from small antral follicles of pre-pubertal unprimed mice and placed in prematuration culture under different CNP-supplemented media conditions. A preliminary analysis showed a dose-dependent effect of CNP on the maintenance of meiotic arrest. A dose of 25nM maintained oocytes under meiotic arrest for 24 h, and this period was extended to 48 h in presence of Estradiol. Analysis of transzonal projections of COCs cultured with CNP indicated that oocyte-cumulus connections were well preserved after the prolonged prematuration culture. Furthermore, CNP medium supplemented with FSH and GDF9 promoted oocyte growth and induced a shift in oocyte chromatin configuration from a predominant disperse- to a condensed configuration. Following IVM, oocytes cultured under CNP were capable to extrude the first polar body at a high rate (around 80%). Blastocyst formation was significantly improved when oocytes were cultured under CNP-supplemented medium containing FSH and GDF9. This study reports for the first time a prolonged prematuration culture system, having CNP as pivotal factor, which can efficiently maintain oocytes retrieved from unprimed prepubertal mice under meiotic arrest while promoting their acquisition of developmental competence.

Concepts: Culture, Meiosis, Polar body, Oocyte

6

Until now, complete ex-vivo spermatogenesis has been reported only in the mouse. In this species the duration of spermatogenesis is 35 days, whereas it is 54 days in the rat and 74 days in the human. We performed long term (until 60 days) cultures of fresh or frozen rat or human seminiferous tubule segments in a bioreactor, made of a hollow cylinder of chitosan hydrogel. Testicular tissues were obtained from 8 or 20 day old male rats, or from adult human subjects having undergone hormonal treatments leading to a near complete regression of their spermatogenesis, before bilateral orchiectomy for gender reassignment. The progression of spermatogenesis was assessed by cytological analyses of the cultures; it was related with a dramatic increase in the levels of mRNAs specifically expressed by round spermatids: Transition protein 1, Transition protein 2 and Protamine 3 in rat cultures. Two to 3.8% of cells were found haploid cells by FISH analysis of human cultures. In this bioreactor, long term cultures of seminiferous tubule segments from (pre)-pubertal rats, or from adult men, allowed the completion of the spermatogenic process, leading to morphologically mature spermatozoa. Further studies will have to address the way of optimizing the yield of every step of spermatogenesis, by adjusting the composition of the culture medium, the geometry and material properties of the chitosan hydrogel bioreactors. Another essential requirement is to assess the quality of the gametes produced ex-vivo by showing their ability to produce normal offspring (rat) or their biochemical normality (human).

Concepts: Protein, Sperm, Spermatozoon, Rat, Meiosis, Sertoli cell, Spermatogenesis, Mouse

5

The peri-conceptual environment represents a critical window for programming fetal growth trajectories and susceptibility to disease however the underlying mechanism responsible for programming remains elusive. This study demonstrates a causal link between reduction of pre-compaction embryonic mitochondrial function and perturbed offspring growth trajectories and subsequent metabolic dysfunction. Incubation of embryos with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), which uncouples mitochondrial oxidative phosphorylation, significantly reduced mitochondrial membrane potential and ATP production in the 8-cell embryo and the number of inner cell mass cells within blastocysts however blastocyst development was unchanged. This perturbed embryo mitochondrial function was concomitant with reduced birth weight in female offspring following embryo transfer which persisted until weaning. FCCP treated females also exhibited increased adiposity at 4 weeks, increased adiposity gain between 4 weeks and 14 weeks, glucose intolerance at 8-weeks and insulin resistance at 14 weeks. Although FCCP treated males also exhibited reduced glucose tolerance their insulin sensitivity and adiposity gain between 4 to 14 weeks was unchanged. This is one of the first studies to demonstrate that reducing mitochondrial function and thus decreasing ATP output in the pre compacting embryo can influence offspring phenotype. This is of great significance as a large proportion of patients requiring assisted reproductive technologies are of advance maternal age or of a high body mass index which have both been independently linked with perturbed early embryonic mitochondrial function.

Concepts: Photosynthesis, Adenosine triphosphate, Enzyme, Mitochondrion, Obesity, Oxidative phosphorylation, Cellular respiration, Embryogenesis

4

The ability to manipulate the genome by adding, removing, or modifying DNA sequences in a sequence specific fashion is essential to studies that investigate the genetic underpinning of physiology. Technology based on the prokaryotic CRISPR (clustered regularly interspersed short palindromic repeats)-Cas9 system is completely revolutionizing genome engineering. The cisgenic and transgenic capabilities of the CRISPR/Cas9 system has very important applications to create animal models and in agriculture and human health. The gene editing can be used to introduce specific base pair changes to recreate important single nucleotide polymorphisms (SNPs). The transgenic approach can be used to create small insertions for introduction of flox sites for downstream conditional deletion strategies using Cre recombinase. Further, it can be used to create large insertions for the knock-in of reporter alleles (LacZ, eGFP, etc.), Cre recombinase, or entire genes to replicate natural copy number variation (CNV). Thus, the CRISPR/Cas9 system should be useful to create designer animals that contain natural SNP and CNV associated with a specific trait or disease. This approach is critical to develop new animal biomedical research models and also agricultural animals with specific desired traits without having to use a complicated breeding scheme for introgression of the SNP or CNV. Introgression for species that have long generation intervals is untenable. Additionally, therapeutics to correct human and animal diseases are another application. In summary, we expect this technology to revolutionize all aspects of science and to provide more sizzling manuscripts in Biology of Reproduction.

Concepts: DNA, Gene, Genetics, Molecular biology, Human genome, Organism, Base pair, Single-nucleotide polymorphism

4

After mating, many female mammals store a subpopulation of sperm in the lower portion of the oviduct, forming a reservoir. The reservoir lengthens sperm lifespan, regulates sperm capacitation, controls polyspermy, and selects normal sperm. It is believed that sperm bind to glycans on the oviduct epithelium to form the reservoir, but the specific adhesion molecules that retain sperm are unclear. Herein, using a glycan array to test 377 glycans for their ability to bind porcine sperm, we found two glycan motifs in common among all glycans with sperm-binding ability: the Lewis X trisaccharide and biantennary structures containing a mannose core with 6-sialylated lactosamine at one or more termini. Binding to both motifs was specific; isomers of each motif did not bind sperm. Further work focused on sialylated lactosamine. Sialylated lactosamine was found abundantly on the apical side of epithelial cells collected from the oviduct isthmus, among N-linked and O-linked glycans. Sialylated lactosamine bound to the head of sperm, the region that interacts with the oviduct epithelium. After capacitation, sperm lost affinity for sialylated lactosamine. Receptor modification may contribute to release from the reservoir so that sperm can move to the site of fertilization. Sialylated lactosamine was required for sperm to bind oviduct cells. Simbucus nigra agglutinin or an antibody specific to sialylated lactosamine with a preference for Neu5Acalpha2-6Gal rather than Neu5Acalpha2-3Gal reduced sperm binding to oviduct isthmic cells, as did occupying putative receptors on sperm with sialylated biantennary glycans. These results demonstrate that sperm binding to oviduct 6-sialylated biantennary glycans is necessary for normal adhesion to the oviduct.

Concepts: Epithelium, Endothelium, Skin