Polycystic ovary syndrome (PCOS) is a complex disorder in which the aetiology is still not explained very well. The PI3K/PTEN (phosphatidylinositol 3-kinase/phosphatase and tensin homolog deleted on chromosome 10) pathway is an important pathway that is involved in many mechanisms, including proliferation, growth and motility. PTEN plays a role in granulosa cell proliferation and regulates the differentiation process. The aim of this study was to investigate the expression levels of Pten and Pik3ca in PCOS mouse models with and without any treatment procedures. Three groups of mouse models, PCOS, a PCOS group with clomiphene citrate treatment, and a PCOS group with the combination of clomiphene citrate, metformin and pioglitazone treatment, were established. Ovarian tissues, which were obtained from these groups and a control group with no PCOS, were embedded in paraffin and RNA was extracted. cDNA was synthesized and real-time PCR was conducted to evaluate the expression levels of Pten and Pik3ca. The results of this study showed that both Pten and Pik3ca genes were expressed in the ovarian tissues from the mouse models. Although one-way analysis of variance results showed that Pten was expressed significantly differently in the samples, individual Student’s t-tests did not show any significantly different expression levels in each group. This study is important as it shows the expression patterns of two genes in PCOS mouse models with different treatment strategies, including clomiphene citrate, metformin and pioglitazone. The results of this study formed the basis of research studies and investigations into different genes within the PTEN pathway, as well as other pathways that are under investigation.

This study aimed to evaluate mRNA levels of angiotensin II (ANG II) receptors (AGTR1 and AGTR2) in caprine follicles and to investigate the influence of ANG II on the viability and in vitro growth of preantral follicles. Real-time polymerase chain reaction (PCR) was used to quantify AGTR1 and AGTR2 mRNA levels in the different follicular stages. For culture, caprine ovaries were collected, cut into 13 fragments and then either directly fixed for histological and ultrastructural analysis (fresh control) or placed in culture for 1 or 7 days in α-minumum essential medium plus (α-MEM+) with 0, 1, 5, 10, 50 or 100 ng/ml ANG II. Then, the fragments were destined to morphological, viability and ultrastructural analysis. The results showed that primordial follicles had higher levels of AGTR1 and AGTR2 mRNA than secondary follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA than their respective cumulus–oocyte complex (COCs). After 7 days of culture, ANG II (10 or 50 ng/ml) maintained the percentages of normal follicles compared with α-MEM+. Fluorescence and ultrastructural microscopy confirmed follicular integrity in ANG II (10 ng/ml). In conclusion, a high expression of AGTR1 and AGTR2 is observed in primordial follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA. Finally, 10 ng/ml ANG II maintained the viability of caprine preantral follicles after in vitro culture.

The present experiments were designed to study the effects of Robertsonian translocations on the efficiency and kinetics of in vitro fertilization and early and advanced embryo development. Spermatozoa from bulls with rob(16;20), rob(1;29) and normal karyotype (A, B and C, respectively) were used. Oocytes were matured, fertilized and cultured by the standard protocol described previously. Twenty-four hours after fertilization, adequate numbers of oocytes were fixed, stained and examined. The development of embryos was evaluated on days 2 (D2), 7 (D7) and 8 (D8) after fertilization. The rate of normally fertilized oocytes was significantly lower (p≤0.01) for bull A than for bulls B and C. However, no significant differences in the kinetics of fertilization were found between bulls A, B and C. The D2 cleavage rate of embryos was significantly lower (p≤0.01) for bull A than for bulls B and C. Both D7 and D8 blastocyst rates for bull A or bull B were significantly lower (p≤0.01 or p≤0.01) than those for bull C. The percentages of both D7 advanced blastocysts and D8 expanded blastocysts were significantly lower (p≤0.01) for bulls A and B than for bull C. In conclusion, for rob(16;20), the efficiency of fertilization was strongly reduced; it resulted in low early and advanced embryo development. On the other hand, for the rob(1;29), neither fertilization nor early embryo development were affected and only advanced embryo development was decreased. But for both translocations, blastocyst formation was significantly delayed.

This study examined the effects of zinc chloride (ZnCl2) and sodium selenite (Na2SeO3) supplementation in maturation medium on in vitro maturation (IVM) rate, oxidative biomarkers and gene expression in buffalo oocytes. Ovaries from a slaughterhouse were aspirated and good quality cumulus–oocyte complexes (COCs) with at least four layers of compact cumulus cells and evenly granulated dark ooplasm were selected. COCs were randomly allocated during IVM (22 h) to one of four treatment groups: (1) control maturation medium (basic medium), or basic medium supplemented with (2) ZnCl2 (1.5 µg/ml), (3) Na2SeO3 (5 µg/l), or (4) ZnCl2 + Na2SeO3 (1.5 µg/ml + 5 µg/l, respectively). Oocytes were denuded after 22 h of IVM in the first four replicates. Specimens were fixed and stained to evaluate the stage of nuclear maturation. The spent medium was collected for biochemical assays of total antioxidant capacity (TAC), malondialdehyde (MDA) and hydrogen peroxide concentrations. A second four replicates were used for COCs for RNA extraction. The expression levels of antioxidant (SOD1, GPX4, CAT and PRDX1), antiapoptotic (BCL2 and BCL-XL) and proapoptotic (BAX and BID) genes were measured. Supplementation with ZnCl2 and Na2SeO3 during IVM increased the ratio of oocytes reaching metaphase II at 22 h, increased TAC and decreased MDA and H2O2 concentrations in the maturation medium (P < 0.05). Moreover, beneficial effects were associated with complementary changes in expression patterns of antioxidative, antiapoptotic and proapoptotic genes, suggesting lower oxidative stress and apoptosis. Supplementation medium with zinc chloride and sodium selenite improves the maturation rate, reduces oxidative stress and increases expression levels of antioxidative and antiapoptotic genes.

Recently, more attention has been raised towards fertility preservation in women with cancer. One option is in vitro maturation (IVM) of the immature oocytes as there is not enough time for induction of an ovarian stimulation protocol. The aim was to compare the IVM laboratory outcomes between stimulated and unstimulated (natural) in vitro fertilization (IVF) cycles. In total, 234 immature oocytes collected from 15 cancer patients who underwent an IVM programme (natural IVM) and 23 IVF cycles with a controlled ovarian hyperstimulation protocol (stimulated IVM) were analyzed. The oocyte morphology, zona pellucida (ZP), and meiotic spindle presence were measured using PolScope technology. Also, the rates of oocyte maturation and fertilization were assessed in both groups. The IVM rate was higher in the stimulated cycle (P < 0.05), but the fertilization rate was insignificant in comparison with unstimulated cycles. There were no significant differences in the spindle visualization and ZP birefringence scoring between the groups (P > 0.05). The oocyte normal morphology was better in the stimulated cycle compared with the natural cycle (P < 0.05). In conclusion, IVM can be recommended for cancer patients as an alternative treatment when there is insufficient time for conventional IVF before chemotherapy initiation.

The lack of preventive policy legislation and the low removal rate of organic pollutants in conventional potabilization treatments lead to some of them being present in drinking water. The problem arises because some of these substances have detrimental effects on human reproduction health, via females, via males or even both. In this work, we established the zebrafish as a bioindicator of these types of substances with the goal of discriminating the effects through three different pathways: male, female or water where the fertilization took place.

For this purpose, four parameters were analysed: fertility rate, hatching rate and survival and abnormalities rates. So, for each parameter two groups were formed, according to whether adult males or females were reared in bottled spring water (Z) or tap water (B) and if the in vitro fertilization took place in water Z or B.

Results revealed a decline in the fertility and hatching rate in water B, due to a water effect. The most plausible explanation could be the presence of substances which affect the micropyle and chorion. Moreover, a decrease in the fertility rate due to an effect over the female was also observed, but in this case by an alteration of the oocyte quality.

Lipopolysaccharide (LPS) endotoxemia has been negatively associated with fertility. This study aimed to investigate the effect of LPS-induced inflammation on gene expression associated with bovine fertility in the uterus and oviduct. Sixteen healthy heifers were divided into two groups. The LPS group (n = 8) received two intravenous (i.v.) injections of 0.5 µg/kg of body weight of LPS with a 24-h interval, and the control group (n = 8) received two i.v. injections of saline solution with the same interval of time. All the animals had the follicular wave synchronized. Three days after the second injection of LPS, all animals were slaughtered and uterine and oviduct samples were collected. Gene expression associated with inflammatory response, thermal and oxidative stresses, oviduct environment quality, and uterine environment quality was evaluated. Body temperature and leucogram demonstrated that LPS induced an acute systemic inflammatory response. In the uterus, the expression of PTGS2 and NANOG genes was downregulated by the LPS challenge. However, no change in expression was observed in the other evaluated genes in the uterus, nor those evaluated in the oviduct. In conclusion, the inflammatory process triggered by LPS did not persist in the uterus and oviduct 3 days after challenge with LPS. Nonetheless, reduction in PTGS2 and NANOG expression in the uterus suggested that, indirectly, LPS may have a prolonged effect, which may affect corpus luteum and endometrial functions.

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.

Determination of optimum sperm requirement in artificial breeding helps to improve the fertilization efficiency, sperm economy and minimize the sacrificing of males for milt. Quantification of sperm cells was done by spectrophotometer at 420 nm followed by haemocytometer counting. Best correlation (r2 = 0.97) was recorded. The minimum number of sperm required for optimal fertilization success in Heteropneustes fossilis was determined. An average fertilization success of 78–93% was recorded at 8 × 103 to 8 × 107 sperm per egg. The optimum contact duration of gametes was 5 minutes. The highest fertilization rate of 98.18% was recorded at 8 × 107 spermatozoa.egg−1.

Early studies have shown that some mouse cumulus–oocyte complexes (COCs) stored at room temperature for 24 h still retained full developmental potential. In this study, we stored denuded mouse oocytes (DOs) at room temperature (25 °C) for 24 h and activated these oocytes with 10 mM SrCl2 or fertilized the oocytes by IVF. We found that nearly half of the DOs stored at room temperature for 1 day can be fertilized normally by IVF and that two foster mothers gave birth to seven pups. Embryos from stored oocytes were cultured in CZB medium with or without 1 μg/ml 17β-estradiol (E2). The numbers of embryo that developed to morula/blastocyst stage after parthenogenetic activation and IVF were significantly increased when E2 was added to the culture (p < 0.05). These results suggest that E2 might improve mouse embryo development in vitro. The birth of seven agouti pups and their healthy growth indicated that the storage of DOs at room temperature for 1 day may be a practical procedure for mammalian reproduction.

Embryos undergo chaotic division and decrease in quality on day 3 with a reduction in the rates of subsequent blastocyst formation. Disordered cleavage causes a deterioration in embryonic quality, here we assessed the relationship between an cleavage model in first mitosis and the chromosomal status of human embryos, and discuss the potential biological and clinical implications for the cleavage model as a single parameter that can be used to assess embryonic quality. Thirty-two infertile couples, with normal karyotypes and who underwent their first IVF cycle were recruited to donate one normal two-cell-stage embryo each for this study between 2019 and 2020. Twenty-eight two-cell embryos underwent preimplantation genetic testing of each blastomere, and four chaotic-division embryos were stained with Hoechst and cultured in a confocal laser-scanning microscopy incubator system. This system showed high specificity and PPV but low sensitivity and NPV using the CM in the prediction of euploidy, indicating that CM could be considered a screening method for embryo selection; additional observational studies using the CM to select transferable embryos are needed before it can be used in clinical practice.

Successful in vitro fertilization (IVF) of all inbred strains of laboratory mice has not yet been accomplished. We have previously shown that a high calcium concentration improved IVF in various inbred mice. However, we also found that in cumulus-free ova of C3H/He mice such IVF conditions significantly increased the deficiency of extrusion of the second polar body (PBII) in a dose-dependent manner (2% at 1.71 mM and 29% at 6.84 mM, P < 0.05) and that PBII extrusion was affected by high calcium levels at 2–3 h post-insemination. While developmental competence of ova without PBII extrusion to blastocysts after 96 h culture was not affected, a significant reduction in the nuclear number of the inner cell mass was observed in blastocyst fertilized under high calcium condition. We also examined how high calcium concentration during IVF affects PBII extrusion in C3H/He mice. Cumulus cells cultured under high calcium conditions showed a significantly alleviated deficient PBII extrusion. This phenomenon is likely to be specific to C3H/He ova because deficient PBII extrusion in reciprocal fertilization between C3H and BDF1 gametes was observed only in C3H/He ova. Sperm factor(s) was still involved in deficient PBII extrusion due to high calcium concentrations, as this phenomenon was not observed in ova activated by ethanol. The cytoskeletal organization of ova without PBII extrusion showed disturbed spindle rotation, incomplete formation of contractile ring and disturbed localization of actin, suggesting that high calcium levels affect the anchoring machinery of the meiotic spindle. These results indicate that in C3H/He mice high calcium levels induce abnormal fertilization, i.e. deficient PBII extrusion by affecting the cytoskeletal organization, resulting in disturbed cytokinesis during the second meiotic division. Thus, use of high calcium media for IVF should be avoided for this strain.

The relative quantity of cyclin B1 was determined during the development of in vitro and in vivo derived porcine 4-cell embryos by western blotting and immunolocalised during the 4-cell stage. After cleavage to the 4-cell stage cyclin B1 localised to the cytoplasm at the 5, 10, 18 and 25 time points and localised to the nucleus 33 h post 4-cell cleavage (P4CC). The relative abundance of cyclin B1 was not significantly different in in vivo or in vitro derived 4-cell stage embryos cultured in the absence of the RNA polymerase inhibitor α-amanitin. Cyclin B1 protein was not detectable in embryos cultured in medium without α-amanitin for 5, 10, 18 or 25 h P4CC followed by culture in medium with α-amanitin to 33 P4CC. These results suggest that the maternal to zygotic transition of mRNA production that occurs at the 4-cell stage of the pig embryo does not result in an increase in cyclin B1 production. In addition, cyclin B1 protein levels remained constant in the absence of embryonic genome activation at the 4-cell stage.

The aim of this study was to evaluate the production of bovine embryos in vitro when supplemented with l-carnitine for 24 h beginning on day 5 (d 5) under two different oxygen tensions (20% or 5%) and the relationship of nitric oxide (NO) in in vitro culture (IVC) medium to embryo development. Cumulus–oocyte complexes (COC; n = 837) were matured in vitro for 24 h and fertilization was performed for 18 h. Zygotes were cultured in vitro for 9 days after in vitro fertilization in synthetic oviductal fluid (SOF) medium with 5% fetal calf serum. At d 5 the plates were assigned to one of four treatment groups: high (20%) or low (5%) O2 tension either with or without the addition of 3.03 mM l-carnitine (High-Cont, High-Lcar, Low-Cont, Low-Lcar). The concentration of NO in the culture medium was evaluated on d 5, d 6 and d 9. On d 7, parts of the embryos were submitted for evaluation of intracellular lipid droplets. The cleavage rate was similar (P > 0.05) between high and low O2 tension and the blastocyst rate was similar in all conditions evaluated. The hatching rate was higher (P < 0.05) for Low-Cont. The NO concentration was higher at d 9 under low O2 tension (P < 0.1). The addition of 3.03 mM l-carnitine between d 5 and d 6 of IVC was not efficient in reducing cytoplasmic lipid content of bovine embryos. Additionally, IVC at a low oxygen tension without l-carnitine promoted better conditions for embryo development. A higher concentration of NO in medium was observed under low O2 tension.

Preovulatory binding of viable spermatozoa in the oviduct isthmus is widely accepted as a preliminary to fertilization, but details of physiological events associated with epithelial binding and release from binding are themselves little understood. Important questions include the potential number, distribution and stability of such sites in the caudal isthmus, whether multiple molecular forms of binding exist within a single-mated individual, and whether some sites are more favourable than others for the maintenance of preovulatory sperm viability. Also to be resolved is whether spermatozoa interact with the first available binding sites in the isthmus, whether spermatozoa from second or subsequent matings bind closer to the site of fertilization, and whether the first spermatozoa entering the oviduct are those that will be released first with impending ovulation. Ideally, future research needs to focus on a fertilizing spermatozoon monitored in vivo and not on spermatozoa destined to remain on or in the zona pellucida or in the lower reaches of the oviduct.

The experimental objective was to determine whether the capability of bovine oocyte plasma membrane to bind sperm changes during in vitro oocyte maturation and fertilisation. Binding was quantified by the intensity of tetramethylrhodamine isothiocyanate (TRITC) fluorescence at the periphery of oocytes following incubation with biotinylated sperm plasma membrane proteins and subsequent incubation with TRITC-avidin. Bovine oocytes were matured in vitro. Sample groups were removed after 0,6 and 22 h, or inseminated and further cultured for 24 or 48 h. Oocytes were denuded of cumulus cells and zona pellucida and co-incubated with 56 μg biotinylated bovine sperm plasma membrane protein for 45 min in 150 μl drops of saline-BSA. Controls were incubated for the same time period in the absence of sperm plasma membrane proteins. All oocytes were rinsed, incubated with TRITC-avidin and subsequently fixed and transferred to mounting medium. Oocytes were scanned with a confocal microscope and analysed using ImageQuant software. The binding of sperm plasma membrane was quantified by integrated fluorescent intensity in standardised ellipses spaced around the plasma membrane of the oocyte. Values are expressed as mean intensity units per 320 pixel ellipse. Binding of sperm plasma membrane continued to increase throughout in vitro oocyte maturation and fertilisation (9051, 24318 and 49953 for 0 and 22 h in vitro matured oocytes and fertilised oocytes, respectively; p = 0.0001). A dramatic decrease in sperm plasma membrane binding to the oocyte plasma membrane was observed in 2-cell embryos (mean intensity = 24477, p = 0.0001). The observed binding was primarily due to the binding of sperm plasma membrane proteins, as control oocytes incubated with TRITC- avidin only were barely visible (integrated fluorescence intensity values ranged from 8 to 3757).

The activity of exoglycosidases in extracts from freshly ejaculated boar and bull spermatozoa with 0.2% Brij-35/2% acetic acid was measured. The results show that β-N-acetylhexosaminidase, β-galactosidase and α-mannosidase are the major glycosidases; much higher levels of activity were found in boar spermatozoa than in bull spermatozoa. When compared on a per spermatozoon basis, the ratios of the activities of β-N-acetylhexosaminidase, β-galactosidase and α-mannosidase in boar spermatozoon relative to those in bull spermatozoon were approximately 13 000:1, 1700:1 and 400:1, respectively. Liberation of these glycosidases from bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) was low, in contrast to liberation of α-mannosidase from boar spermatozoa previously found by the same means. The possibility that the exoglycosidases present in large amounts in boar spermatozoa play a role in the process of binding to the zona pellucida glycoprotein of the egg is discussed.

Balanced reciprocal translocation carriers are usually phenotypically normal. Although the reproductive risk of these carriers varies, they generally have a lower chance to produce normal or balanced gametes. Preimplantation genetic diagnosis (PGD) is offered to these patients to increase their chances of becoming pregnant by selecting a balanced embryo for transfer. This study aimed to analyse the development and the PGD outcome of the embryos obtained from reciprocal translocation carriers focusing on ones with chromosome 10 rearrangements. In total, 27 reciprocal translocation carriers underwent 31 cycles of PGD. PGD was performed using multicolour fluorescence in situ hybridisation for 298 embryos and of these 136 were obtained from couples carrying translocations involving chromosome 10 rearrangements. Carriers of translocations involving chromosome 10 rearrangements have a lower chance of producing normal or balanced embryos compared with the carriers with other rearrangements. The development of embryos obtained from the patients with chromosome 10 rearrangements was impaired and only a limited number of embryos developed to the blastocyst stage.

Algae were supplied continuously to four-armed pluteus larvae of the sea urchin Hemicentrotus pulcherrimus until the adult rudiment reached stage g, which is the initial stage of rudiment formation (Chino et al., 1994) After stage g, one group of larvae was reared without the addition of algae for comparison of the development of the adult rudiment with that in larvae given algae. Three days later, only 9.1 ± 0.3% of larvae without algae had reached stage j, while 76.8 ± 0.6% of larvae with algae had reached a stage beyond j, which indicates the formation of the complete adult rudiment.

When larvae at rudiment stage g were reared in a medium supplemented with T4 or its derivatives, such as T3, 3,3′,5′-L-triiodothyronine (rT3) or triiodothyropropionine (Tp3), in place of algae, the adult rudiment developed in a dose-dependent manner. T4 was the most effective and induced formation of the adult rudiment in more than 70% of specimens at 1 nM and in almost 100% at 100 nM. T3 was one-tenth as effective as T4. Other derivatives were still less effective. On the other hand, casein, ovalbumin, tyrosine and a mixture of 20 amino acids had no effect on the development of larvae and adult rudiments, suggesting that they were not available as nutrients and sources of thyroid hormones.