In zebrafish chimaerism experiments, the cell injection can involve intra-embryonic cell lyses by osmolar effects. Moreover, the donor cells can be injured during manipulation due to osmolar changes into the transplant pipette. Therefore, the present study aimed to assess the effects of manipulation medium osmolarity on embryonic survival and donor cell viability.

In Experiment I, 0.1 μl to 0.15 μl approximately of an isosmolar solution (300 mOsm) was injected into recipient embryos, which were kept at 300 (E1) or 30 mOsm (E2). Survival at day 1 was significantly higher in the E2 group than in E1 (E1: 68% vs E2: 81%, p < 0.05), but after 5 days embryo survival in the E1 group was slightly higher. In Experiment II, donor cells from zebrafish embryos were exposed (or not) to a possible osmolarity change (inner pipette medium: 300 mOsm vs external medium: 30 or 300 mOsm) using two different micropipette outer diameters, 40–50 and 60–70 μm. Cell mechanical damage was detected in the 40–50 μm pipette (p < 0.05), but not by the handling medium osmolarity. Results recommend the use of a 300 mOsm manipulation medium and bore-sized pipettes adjusted as closely as possible to the donor cell size.

Abnormalities in the zona pellucida (ZP) adversely affect oocyte maturation, embryo development and pregnancy outcomes. However, the assessment of severity is challenging. To evaluate the effects of different degrees of ZP abnormalities on embryo development and clinical outcomes, in total, 590 retrieval cycles were scored and divided into four categories (control, mild, moderate and severe) based on three parameters: perivitelline space, percentage of immature oocytes and percentage of oocytes with abnormal morphology. As the severity of abnormal ZP increased, both the number of retrieved oocytes and mature oocytes decreased. The fertilization rate did not differ significantly among groups. The rates of embryo cleavage and day-3 high-quality embryos in the mild group and the moderate group did not vary significantly between the two groups but were significantly higher than those in the severe group. The blastulation rates of the abnormal ZP groups were similar; however, they were lower than those of the control group. Moreover, the cycle cancellation rate of the severe abnormal ZP group was as high as 66.20%, which was significantly higher than that of the other three groups. Although the rates of cumulative clinical pregnancy and live births were lower than those in the control group, they were comparable among the abnormal ZP groups. There were no differences in the neonatal outcomes of the different groups. Together, ZP abnormalities show various degrees of severity, and in all patients regardless of the degree of ZP abnormalities who achieve available embryos, there will be an opportunity to eventually give birth.

Glyphosate is a universal herbicide with genital toxicity, but the effect of glyphosate on oocytes has not been reported. This study aimed to evaluate the effect of glyphosate (0, 10, 20, 50 and 100 mM) on bovine oocyte in vitro maturation. We showed that 50 mM glyphosate adversely affects the development of bovine oocytes. Exposure of oocytes to 50 mM glyphosate caused an abnormal reduction in oxidative (redox) levels compared with that in the control group, with a significantly higher reactive oxide species level (P < 0.05) and significantly lower glutathione (GSH) expression (P < 0.05). Additionally, the mRNA levels of antioxidant genes (SOD1, SOD2, SIRT2, SIRT3) and the mitochondrial membrane potential (MMP) were significantly reduced (P < 0.05). Furthermore, treatment with 50 mM glyphosate-induced apoptosis, and the mRNA levels of the apoptotic genes Caspase-3 and Caspase-4 were significantly higher than those in the control group (P < 0.05); however, the mRNA level of BAX was significantly higher than that in the control group (P < 0.01). Additionally, the mRNA levels of the anti-apoptotic genes Survivin and BCL-XL were significantly lower than those in the control group (P < 0.05), and oocyte quality was adversely affected. Together, our results confirmed that glyphosate impairs the quality of oocytes by promoting abnormal oocyte redox levels and apoptosis.

To highlight the ultrastructural features of transformation occurring with germplasm-related structures (GPRS), the spermatogenic cells of Xenopus laevis were studied by transmission electron microscopy and quantitative analysis. It was determined that in spermatogonia and spermatocytes, the compact germinal granules underwent fragmentation into particles comparable with inter-mitochondrial cement (IMC). Fragments of IMC agglutinated some cell mitochondria and resulted in the creation of mitochondrial clusters. Clustered mitochondria responded with loss of their membranes that occurred by the twisting of membranous protrusions around themselves until multi-layered membranes were formed. The mitochondrial affinity of multi-layered membranes was proven by an immunopositive test for mitochondrial dihydrolipoamide acetyltransferase. As a consequence of mitochondrial membrane twisting, the naked mitochondrial cores appeared and presumably underwent dispersion, which is the terminal stage of GPRS transformation. As no GPRS were observed in spermatids and sperm, it was assumed that these structures are functionally assigned to early stages of meiotic differentiation.

High rates of infertility in type 2 diabetic (T2DM) men have led to attempts to understand the mechanisms involved in this process. This condition can be investigated from at least two aspects, namely sperm quality indices and epigenetic alterations. Epigenetics science encompasses the phenomena that can lead to inherited changes independently of the genetics. This study has been performed to test the hypothesis of the relationship between T2DM and the epigenetic profile of the sperm, as well as sperm quality indices. This research included 42 individuals referred to the infertility clinic of Royan Institute, Iran in 2019–2021. The study subjects were assigned to three groups: normozoospermic non-diabetic (control), normozoospermic diabetic (DN) and non-normozoospermic diabetic (D.Non-N). Sperm DNA fragmentation was evaluated using the sperm chromatin structure assay technique. The global methylation level was examined using 5-methyl cytosine antibody and the methylation status in differentially methylated regions of H19, MEST, and SNRPN was assessed using the methylation-sensitive high-resolution melting technique. The results showed that the sperm global methylation in spermatozoa of D.Non-N group was significantly reduced compared with the other two groups (P < 0.05). The MEST and H19 genes were hypomethylated in the spermatozoa of D.Non-N individuals, but the difference level was not significant for MEST. The SNRPN gene was significantly hypermethylated in these individuals (P < 0.05). The results of this study suggest that T2DM alters the methylation profile and epigenetic programming in spermatozoa of humans and that these methylation changes may ultimately influence the fertility status of men with diabetes.

The live birth rate following embryo transfer is comparable between spontaneous, stimulated and artificial cycles. However, the pregnancy loss rate appears elevated with hormonal therapy, possibly due to luteal insufficiency. This study aimed to determine whether the serum progesterone level on transfer day differed according to the endometrial preparation method for frozen embryo transfer (FET). Twenty spontaneous cycles (SC), 27 ovarian stimulation cycles (OS) and 65 artificial cycles (AC) were retrospectively studied from May to December 2019 in a single French hospital. The primary endpoint was the level of serum progesterone on the day of FET between the three endometrial preparation methods. The mean serum progesterone level on transfer day was 29.47 ng/ml in the OS group versus 20.03 ng/ml in the SC group and 14.32 ng/ml AC group (P < 0.0001). Progesterone levels remained significantly different after logistic regression on age and anti-Müllerian hormone (AMH) level. There was no significant difference in demographic and hormone characteristics (age, body mass index, embryo stage of embryo, type of infertility, basal follicle stimulating hormone, luteinizing hormone, estradiol and AMH levels), endometrial thickness, number and type of embryos transferred, duration of infertility, pregnancy rate, live birth rate and pregnancy loss rate. No difference was found in serum progesterone levels between clinical pregnancy with fetal heartbeat and no clinical pregnancy (no pregnancy or pregnancy loss, 17.49 ng/ml vs 20.83 ng/ml, respectively, P = 0.07). The lower serum progesterone level found on FET day in the AC group should be further investigated to see whether this difference has a clinical effect on the live birth rate.

Protease activities have been recognised as important elements in controlling the composition of the extracellular matrix. Regulated remodelling of the matrix is required for a number of physiological processes including embryonic development. Excessive and unregulated remodelling has been associated with a number of pathological conditions including the metastatic phenotype of malignant cancer (Kim et al., 1998). We have begun a search for protease activities which utilise components of the sea urchin extracellular matrix as substrates. We have identified and purified a 41 kDa protease which is present in the sea urchin egg and embryo. This species possesses a non-specific gelatin-cleavage activity as well as a collagen cleavage activity which appears to be specific for echinoderm collagen (Mayne & Robinson, 1996, 1999).

The 41 kDa collagenase/ gelatinase was inhibited by EGTA and reactivated by calcium. The calcium-concentration dependence for reactivation indicated an apparent kd of 3.7 mM and was coincident with the binding of 80 moles calcium/mole of protein. These results are interpretable in terms of the high concentration of calcium (10 mM) present in seawater. In addition to calcium, seawater also contains 50 mM magnesium. The substantial amounts of calcium bound to the 41 kDa protease suggest the existence of binding sites with both low affinity and specificity for binding metal ions. To determine whether high concentrations of magnesium could influence the interaction of calcium with the 41 kDa species we used both qualitative and quantitative gelatin-cleavage assays to examine protease activity in the presence of both calcium and magnesium.

The role of hyaluronic acid (HA) as a ‘physiologic selector’ is also well recognized in vitro: it has been demonstrated that spermatozoa that bind to immobilized HA in vitro are those having completed their plasma membrane remodelling, and cytoplasmic and meiotic maturation. Sperm selection using HA has been expected to increase the implantation rate in intracytoplasmic sperm injection (ICSI) cycles. This work was designed to evaluate an alternative product for slowing sperm motility that contains HA and measures its outcomes: fertilization rate, embryo quality, and implantation and pregnancy rates. The present study found a positive drift in embryo quality that was statistically significant in the study group (SpermSlow™-ICSI) with teratozoospermia compared with PVP-ICSI in the same group. There were differences in the pregnancy rate (statistically insignificant in normozoospermia, asthenozoospermia, oligozoospermia, and teratozoospermia) in the SpermSlow-ICSI group compared with PVP-ICSI. The HA-ICSI technique in assisted reproduction technology (ART) is an important way to improve fertilization rate, embryo quality, and pregnancy rate.

The heterodimeric sperm-coating protein CFS was previously localised on the middle-piece region of rat spermatozoa by anti-CFS rabbit antibodies. CFS-immunorelated antigens were detected in the secretion of the water buffalo seminal vesicle by protein electrophoresis and Western blotting. Spermatozoa from buffalo epididymal cauda were incubated with the rat antigen and, upon immunostaining with anti-CFS antibodies and goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated IgGs, CFS was found attached on both the post-acrosomal region and the tail. Indirect immunofluorescence analysis permitted the localisation of CFS-related antigens on the same domains of buffalo ejaculated spermatozoa. These results suggest that the buffalo antigens not only share some epitopes with the homologous rat antigen but may also have some of its functional properties. Ejaculated spermatozoa were capacitated in vitro and then assayed for their content of CFS-like antigens. An inverse relationship was found between the levels of capacitation and the amounts of antigens detected, thus suggesting that the in vitro treatment was effective at removing CFS-related proteins from the cell surface. Titration of these proteins to proteins to monitor plasma membrane changes during sperm manipulation or to evaluate sperm quality is proposed.

The injection of spermatozoa into mouse, human and rabbit oocytes at specific times and positions can result in different rates of viable embryo development. However, it is not clear how the timing and position of round spermatid injection (ROSI) affect pronucleus (PN) formation and blastocyst development of mice. First, we determined the changes in relative position of the first polar body and the spindle, carried out ROSI from 11.5 to 13 h post-hCG administration, then activated by Sr2+, and finally compared the development of ROSI zygotes, including the formation of pronuclei and development of blastocyst. Between 11.5 and 13 h post-hCG administration, the rate of 2PN formation by ROSI at 3 o'clock was the highest among all treated oocytes. Moreover, the blastocyst rate of zygotes with two pronuclei (2PN) was up to 27.41%. These results suggest that the time and position of ROSI can significantly influence the formation of 2PN, that the rates of 2PN formation are closely correlated with blastocyst formation and that the formation of 2PN is necessary for later embryo development.

Sesquiterpene lactones (STLs) are a large and structurally diverse group of plant metabolites generally found in the Asteraceae family. STLs exhibit a wide spectrum of biological activities and it is generally accepted that their major mechanism of action is the alkylation of the thiol groups of biological molecules. The guaianolides is one of various groups of STLs. Anti-tumour and anti-migraine effects, an allergenic agent, an inhibitor of smooth muscle cells and of meristematic cell proliferation are only a few of the most commonly reported activities of STLs. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under stimulus with progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. There are previous records of the inhibitory effect of dehydroleucodin (DhL), a guaianolide lactone, on the progression of meiosis. It has been also shown that DhL and its 11,13-dihydroderivative (2H-DhL; a mixture of epimers at C-11) act as blockers of the resumption of meiosis in fully grown ovarian oocytes from the amphibian Rhinella arenarum (formerly classified as Bufo arenarum). The aim of this study was to analyze the effect of four closely related guaianolides, i.e., DhL, achillin, desacetoxymatricarin and estafietin as possible inhibitors of meiosis in oocytes of amphibians in vitro and discuss some structure–activity relationships. It was found that the inhibitory effect on meiosis resumption is greater when the lactone has two potentially reactive centres, either a α,β–α′,β′-diunsaturated cyclopentanone moiety or an epoxide group plus an exo-methylene-γ-lactone function.

Sperm-mediated gene transfer (SMGT) is a simple, fast, and economical biotechnological tool for producing transgenic animals. However, transgene expression with this technique in bovine embryos is still inefficient due to low uptake and binding of exogenous DNA in spermatozoa. The present study evaluated the effects of sperm membrane destabilization on the binding capacity, location and quantity of bound exogenous DNA in cryopreserved bovine spermatozoa using Triton X-100 (TX-100), lysolecithin (LL) and sodium hydroxide (NaOH). Effects of these treatments were also evaluated by intracytoplasmic sperm injection (ICSI)-SMGT. Results showed that all treatments bound exogenous DNA to spermatozoa including the control. Spermatozoa treated with different membrane destabilizing agents bound the exogenous DNA throughout the head and tail of spermatozoa, compared with the control, in which binding occurred mainly in the post-acrosomal region and tail. The amount of exogenous DNA bound to spermatozoa was much higher for the different sperm treatments than the control (P < 0.05), most likely due to the damage induced by these treatments to the plasma and acrosomal membranes. Exogenous gene expression in embryos was also improved by these treatments. These results demonstrated that sperm membrane destabilization could be a novel strategy in bovine SMGT protocols for the generation of transgenic embryos by ICSI.

Bufo arenarum oocytes are oviposited surrounded by jelly coats, one component of the extracellular matrix required for fertilization. The secretion, released to the oviductal lumen, was analysed by SDS-PAGE. The coomassie blue staining evidenced an electrophoretic pattern with molecules ranging between 300 and 19 kDa that showed variations in their secretion profiles during the sexual cycle. In the preovulatory period the densitometric analysis showed the presence of nine peaks with marked predominance of the 74 kDa molecule. Once ovulation has occurred, the jelly coats become arranged around the oocytes during their transit throughout the oviductal pars convoluta (PC), revealing the addition of three proteins only observed during this period, which suggests a differential secretion. Some of these proteins could not diffuse under any extraction treatment, indicating for them a structural or in situ function. Proteins of low molecular mass diffused totally while others showed a partial diffusing capacity. After ovulation a marked decrease in the relative amount of all the proteins released to the lumen, especially the 74 kDa protein, could be detected. During this period, unlike the other stages of the sexual cycle, a differential secretion pattern was observed along the PC. The histochemical analysis performed during the ovulatory period showed the presence of glycoconjugates including both acidic and neutral groups. The present results are in agreement with previous ultrastructural and histochemical studies that describe the role of Bufo arenarum jelly coats in fertilization.

To study the role of orexin A in the reproductive regulation of Mongolian sheep, ovine ovarian granulosa cells were cultured in vitro. The cells were divided into groups after luteinization, the experimental group was given orexin A and the transcriptome was sequenced together with that of the control group. The different genes related to reproduction were screened out. qRT-PCR, western blot and enzyme-linked immunosorbent assay (ELISA) were used to verify the selected genes and detect the effect on progesterone secretion. In total, 123 differentially expressed genes were obtained by sequencing. Six genes with high expression related to reproduction (PRRT2, ABCG1, SOX4, TBX3, ID1 and ATP8) were screened. The results of qRT-PCR were consistent with those of sequencing; western blot and ELISA were used to verify the protein levels of steroidogenic acute regulatory protein (StAR) and its related PRRT2 and ABCG1, and to detect their effect on progesterone secretion. Validation results were consistent with those of qRT-PCR and sequencing. The experimental group was given orexin A and compared with the control group. Expression of PRRT2 protein was significantly increased (P < 0.05), ABCG1 protein expression was significantly decreased (P < 0.05), StAR expression was significantly increased (P < 0.05), and progesterone secretion was significantly increased (P < 0.05). The results showed that orexin A promoted the expression of StAR by upregulating PRRT2 and downregulating ABCG1, therefore affecting secretion of progesterone. Gene expression characteristics of orexin A affecting progesterone secretion were preliminarily explored; this study provides a theoretical basis for further study on signalling pathways and reproductive regulation in Mongolian sheep.

The aim of the study was to investigate the continuous changing pattern of H4K12 acetylation, and the expression levels of histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs) in mouse oocytes during meiosis and after parthenogenetic activation (PA). The immunofluorescence results showed hyperacetylation of lysine-12 on histone H4 (H4K12) in the germinal vesicle (GV) oocytes that then decreased during germinal vesicle breakdown (GVBD), and disappeared in metaphase II (MII). However, it reappeared in the early 1-cell embryos derived after 4 h of PA. The expression levels of some selected HATs and HDACs also validated the changing pattern of H4K12 acetylation during meiosis and PA. In conclusion, H4K12 is deacetylated in GVBD and MII, and re-hyperacetylated after PA.

Fresh cytosols extracted from unfertilised eggs of Xenopus laevis contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca2+ and is designated primary CSF (1°CSF). During storage of Ca2+-containing cytosols at 2°C, a stable CSF activity appears which is designated secondary CSF (2°CSF). In the present study, we report that 2°CSF activity can be induced in cytosols extracted from stage VI oocytes, unfertilised eggs, electrically activated eggs or blastulae, in the presence of Ca2+. Both the intensity and the rate of 2°CSF development are dependent on the concentration of Ca2+ ions added to the cytosol. At Ca2+ concentrations of 5–10 mM, 2°CSF activity reaches a maximum in about 7 days. Secondary CSF is relatively resistant to heat but loses all activity after 5 min at 70°C. When stored at −80°C, 2°CSF activity remains detectable for about 6 weeks. Cytological observations show that blastomeres arrested by microinjection of 2°CSF developed in cytosols of unfertilised eggs, activated eggs or blastulae contain metaphase chromosomes embedded in a bipolar spindle that has no asters developed at its poles. In contrast, blastomeres arrested by 2°CSF in cytosols of stage VI oocytes contain condensed chromosomes but no spindle is formed. The mechanisms of the development of 2°CSF in Ca2+-containing cytosols and its mode of action are discussed.

There is no information about the characteristics of early cleavage in the Patagonian blennie (Eleginops maclovinus), which can be used as a diagnostic tool for embryo quality. The purpose of this investigation, therefore, was to characterize the first blastomeres of E. maclovinus morphologically. Of a ‘pool’ of incubated eggs at 10.7 ± 0.5°C, 100 microphotographs of blastodiscs were extracted at different incubation periods from 0.25 to 5 h after fertilization and analyzed. Blastodiscs taken at 3.5, 4.0 and 5.0 h were characterized and classified into symmetric or asymmetric groups according to their morphology. The proportions of length (L) and width (W) of each blastomere were determined to establish its symmetry. Additionally, 20 microphotographs of blastodiscs of normal appearance were analyzed morphologically (control blastodisc: CB) and compared other blastodiscs (4.0 and 5.0 h). The results showed that before fertilization oocytes presented a somehow turgid aspect (maximum average diameter of 987 ± 41 µm) and after fertilization and hydration, their diameter increased to 1001.5 ± 11 µm (but not statistically significant) and presented a spherical shape. First cleavage ends after 3.5 h of development, forming two blastomeres 467 ± 45 μm length (L) and 328 ± 21 μm width (W) with a L/W ratio of 1.43 ± 0.19. The second cleavage ends after development at 4.5 h forming four blastomeres 238 ± 65 μm length and 227 ± 65 μm width with a ratio L/W of 1.06 ± 0.09. Five categories were identified during the blastomere characterization: 70% normal or symmetric; 8% with odd numbers of blastomeres; 6% unequal; 6% ‘pie shaped’ and 10% amorphous.

The glycoprotein envelope surrounding the Bufo arenarum egg exists in different functional forms. Conversion between types involves proteolysis of specific envelope glycoproteins. When the egg is released from the ovary, the envelope cannot be penetrated by sperm. Conversion to a penetrable state occurs during passage through the pars recta portion of the oviduct, where oviductin, a serine protease with trypsin-like substrate specificity, hydrolyzes two kinds of envelope glycoproteins: gp84 and gp55. The nucleotide sequence of a 3203 bp B. arenarum oviductin cDNA was obtained. Deduced amino acid sequence showed a complete open reading frame encoding 980 amino acids. B. arenarum oviductin is a multi-domain protein with a protease domain at the N-terminal region followed by two CUB domains and toward the C-terminal region another protease domain, which lacked an active histidine site, and one CUB domain. Expression of ovochymase 2, the mammalian orthologous of amphibian oviductin, was assayed in mouse female reproductive tract. Ovochymase 2 mRNA was unnoticeable in the mouse oviduct but expression was remarkable in the uterus. Phylogenetic relationship between oviductin and ovochymase 2 opens the possibility to understand the role of this enzyme in mammalian reproduction.

Parthenogenetic activation of oocytes is a helpful tool to obtain blastocysts, of which the inner cell mass may be used for derivation of embryonic stem cells. In order to improve activation and embryonic development after parthenogenesis, we tried to use sperm injection and subsequent removal of the sperm head to mimic the natural Ca2+ increases by release of the oocyte activating factor. Visualization of the sperm could be accomplished by Hoechst staining and ultraviolet (UV) light irradiation. To exclude negative effects of this treatment, we examined toxicity on activated mouse oocytes. After activation, oocytes were incubated in Hoechst 33342 or 33258 stain and exposed to UV irradiation. The effects on embryonic development were evaluated. Our results showed that both types of Hoechst combined with UV irradiation have toxic effects on parthenogenetically activated mouse oocytes. Although activation and cleavage rate were not affected, blastocyst formation was significantly reduced. Secondly, we used MitoTracker staining for removal of the sperm. Sperm heads were stained before injection and removed again after 1 h. However, staining was not visible anymore in all oocytes after intracytoplasmic sperm injection. In case the sperm could be removed, most oocytes died after 1 day. As MitoTracker was also not successful, alternative methods for sperm identification should be investigated.

In Scutigera coleoptrata, double spermatogenesis (macro and micro germinal cells) was reinvesti gted by means of both image analysis and electron microscopy. Image analysis of the cell-lines showed no differences in DNA content between the two types of spermatogesis.The only difference found, with the exception of the size and the related number of organelles, was that in the macro-cell line the chromatin was well dispersed and no condensed, which is the opposite of what was observed in the macro-cell line. It is asumed that the level of activity related to this condensation process is at the origin of the two pathways of development.