Treatment with follicle-stimulating hormone (FSH) and testosterone (T2) and their combination have been observed to be influential on ovarian follicles of 1-day-old mice ovaries cultured for 8 days. Given that extension of the culture period could positively impact the development of follicles in cultured ovaries, the present study was conducted to evaluate the main and interaction effects of FSH by T2 on the development of ovarian follicles in 1-day-old mice ovaries cultured for 12 days. One-day-old mice ovaries were initially cultured with base medium for 4 days; thereafter, different hormonal treatments were added to the culture media, and the culture was continued for 8 additional days until day 12. Ovaries were collected for histological and molecular assessments on day 12. The greatest activation of primordial follicles and progression of activated follicles to the preantral stage was detected in ovaries treated with the combination of FSH and T2 (P < 0.05). This positive effect on the morphology of ovarian follicles was accompanied by upregulation of Pi3k, Gdf9, Bmp15, Cx37 and Fshr in the ovaries cultured with the combination of FSH and T2 (P < 0.05). Nonetheless, treatment with FSH and T2 led to a diminished proportion of intact follicles (P < 0.05), even though Bax/Bcl2 gene expression ratio, as an apoptotic index, was less in hormone-treated ovaries (P < 0.05). In conclusion, the combination of FSH and T2 could improve the activation of primordial follicles and the growth of activated follicles towards the preantral stage. This positive effect of FSH plus T2 appeared to be at least partly mediated through the upregulation of Pi3k and oocyte-derived growth factors including Gdf9 and Bmp15.

Globally, numerous infertile couples have been assisted by extensive research on mammalian fertilization and the rapid development of Assisted Reproductive Technology (ART). However, 5%–15% of the couples that are selected for in vitro fertilization (IVF) experience a total fertilization failure (TFF), where no zygotes develop despite oocytes and semen parameters appear to be normal. Notably, an essential early event in fertilization is the binding of spermatozoa to the oocyte’s external envelope, which followed by the spermatozoa-oocyte fusion. Meanwhile, oocyte activation is a crucial cellular process necessary to block polyspermy and start the development of the zygote. Improper membrane fusion of gametes has been demonstrated to be one of the mechanisms of TFF. Moreover, considering the large amount of research on sperm proteins in recent years, thus in this review, we characterize the role and molecular mechanisms of sperm proteins in the three key processes of gamete adhesion and fusion and oocyte activation, which would provide a comprehensive understanding of the role of sperm proteins in fertilization in mammals and a favourable reference for future studies in assisted reproduction due to FF.

Rainbow trout (Oncorhynchus mykiss) is a promising cultivable fish species with significant potential for expansion. As a cold-water fish belonging to the Salmonidae family, it requires an optimal temperature range of 10–15°C for optimal growth. This study explores a method for producing sterile rainbow trout with maximum survival rates by using heat shock treatment to enhance growth characteristics and improve aquaculture practices. A control group and four heat shock treatments were given at 26°C and 28°C for 10 min, applied 15 and 20 min after the mixing of eggs and milt, using a water bath. Among the treated groups, the highest fertilisation, hatching and yolk sac absorption rates were 90.3 ± 0.3%, 81.8 ± 0.8% and 83.9 ± 0.5%, respectively. The highest triploidy rate of 76.6 ± 3.3% was observed with a heat shock at 28°C, 20 min after fertilisation. In contrast, none of the fish from the control group were triploids. The control group demonstrated higher survival rates at fertilisation (93.1 ± 0.4%), hatching (84.2 ± 0.4%) and complete yolk sac absorption (86.2 ± 0.5%) compared to the heat-shocked groups. The diploid and triploid chromosome numbers in rainbow trout were determined to be 2n = 60 and 3n = 91, respectively. This study confirms that heat shock treatment can effectively induce triploidy in rainbow trout, with significant variations in triploidy rates depending on the temperature and timing of the shock. While heat shock can enhance the production of sterile fish, it is essential to balance the treatment parameters to maintain high survival rates. These findings contribute to the optimisation of triploidy induction techniques and support the advancement of aquaculture practices by improving the growth, management and survival rates of rainbow trout which could significantly benefit aquaculture efficiency and sustainability.

Introduction: Long non-coding RNAs (lncRNAs) are a subset of RNA molecules that have been shown to be involved in gene regulation. A lot of different pathways are involved during gametogenesis and any disturbance to these pathways may have a derogatory impact on producing a haploid gamete and thus a euploid embryo. Steroidogenesis pathway plays a crucial role in gametogenesis. The purpose of this work was to quantify the levels of lnc-CYP11A1-1 and RP11573D15.8 expression levels in aneuploid and euploid embryos. Materials and methods: A total of 20 surplus human embryos, of which 10 euploid and ten aneuploid embryos, were collected from an IVF centre. The expression levels of two lncRNAs, which have been hypothesized to regulate expression of CYP11A1, were evaluated in these embryos. RNA was extracted and used to synthesize cDNA for the experiments. Real-time polymerase chain reaction was performed to evaluate the expression levels of each lncRNA in aneuploid and euploid embryos, respectively. Results and discussion: This study shows that lnc-CYP11A1-1 was more expressed in aneuploid than in euploid embryos. RP11-573D15.8 is expressed more in aneuploid embryos than in euploid ones. The results for RP11-573D15.8 were statistically significant with a p-value of 0.02 (less than the standard threshold of p 0.05), whereas the results for lnc-CYP11A1-1 were not statistically significant with a p-value of 0.07 (greater than the standard threshold of p 0.05). Thus, the result of this study demonstrates that lncRNAs may have a role in gametogenesis and formation of aneuploid gametes.

This study explores the efficacy of a novel microfluidic device in isolating rheotactic sperm and assesses their advantages compared with other motile sperm. Two microfluidic devices were used in this study: the microfluidic device we designed to separate sperm based on rheotaxis and a simple passive microfluidic device. We compared the results with the density gradient centrifugation technique. Sperm attributes including concentration, morphology, viability and motility were assessed using related procedures. Statistical analyses were conducted using one-way analysis of variance. Results showed differences in sperm concentration, motility, morphology and vitality using different sperm separation techniques. The sperms separated using our microfluidic device demonstrated the highest motilities, normal morphology percentages and higher sperm vitality but significantly lower sperm concentrations. These findings suggest the potential of our microfluidic design in enhancing sperm quality. Our findings are in agreement with previous research, emphasizing the capability of microfluidics in enhancing sperm quality. Specifically, our designed microfluidic device exhibited exceptional efficacy in isolating highly motile sperm, a critical factor for successful fertilization.

Obesity, a global health issue, is associated with numerous diseases and has been shown to affect male reproductive health by inducing endocrine hormonal changes, chronic inflammation, oxidative stress and epigenetic alterations in reproductive cells. This study investigates the impact of obesity on testicular gene expression across mice, monkeys and humans, identifying 730 conserved testis-specific genes. High-fat diet-induced obesity upregulates GNG5, INHA, MSH5, SLC30A8 and SLC7A4 in testes, suggesting their potential as regulatory targets in testicular damage associated with obesity. Single-cell analysis reveals species-conserved expression patterns of SLC7A4 in Sertoli cells and SLC30A8 in SPG cells. It also confirmed that SLC30A8 and SLC7A4 were significantly upregulated in the testes of spontaneously obese mice. The findings highlight the potential of these genes as regulatory targets in obesity-related testicular dysfunction, providing insights into male reproductive health impairments caused by obesity.

Immunological castration can be an alternative to traditional surgical castration. The active immunization against GnRH or kisspeptin has a castrating effect. To date, the fusion protein vaccine of combination with GnRH and kisspeptin have not been studied. Thus, the present study will develop a GnRH6-kisspeptin vaccine by genetic engineering method and investigate its immunocastration effect in male rats. Twenty 20-day-old male rats were randomly divided into two groups: the control group (n=10) and the immunization group (n=10). The initial immunization took place at week 0 followed by three booster doses administered intervals. The control group received an equivalent dose of white oil adjuvant. Orbital blood samples were collected at various time points following the initial immunization, at 0, 2, 4, 6, 8, 10 and 12 weeks, respectively. The entire left testis was weighed and its volume measured at week 12. Samples from the right testis were obtained for histological analysis. Serum levels of GnRH and kisspeptin antibodies, as well as testosterone levels were determined using ELISA. The results showed that the serum levels of GnRH and kisspeptin antibody titres of the immunized rats were significantly higher compared to the control group (P<0.05). Additionally, the testosterone concentration was effectively reduced following the intensified immunization. The testes of the immunized group exhibited a reduction in size and a significant decrease in the number of spermatogonia in the testicular tissue compared to the control group (P<0.05). These data indicate that the recombinant GnRH6-kisspeptin protein effectively induced immunological castration in rats.

In cattle, maternal metabolic health has been suggested to influence oocyte and embryo quality. Here, we examined whether maternal liver abnormalities affected in vitro oocyte maturation by screening meiotic maturation, spindle morphology, actin filaments, and lysosomes. In oocytes from the abnormal liver group, the maturation rate (80.2%) was significantly lower compared to a control group with healthy livers (90.8%; P < 0.05). Mean spindle area in oocytes of the abnormal group (50.4 ± 3.4 μm2) was significantly larger than in the control (40.8 ± 1.6 μm2; P < 0.05). Likewise, mean spindle width in the abnormal group (8.8 ± 0.3 μm) was significantly larger than in the control group (7.8 ± 0.2 μm; P < 0.05). The proportion of cells with correctly aligned chromosomes in the abnormal group (48.0%) was significantly lower than in the control (78.3%; P < 0.05). The number of cortical actin filaments in mature oocytes of the abnormal group (299.3 ± 3.7) was significantly lower than in the control (314.7 ± 3.2; P < 0.05). The number of lysosomes in mature oocytes of the abnormal group (1363.6 ± 39.0) was significantly higher than in the control (1123.4 ± 26.3; P < 0.05). In conclusion, our findings indicate that the quality of in vitro matured oocytes is lower in cattle with liver abnormalities than in healthy cattle.

Recently, the World Health Organization recommendation for abstinence time for semen analysis has been challenged in some studies and many of them have supported the advantages of a second short abstinence ejaculation. More evidence is needed to approve this for clinical use. This study aimed to compare the average routine abstinence time (2–7 days) with the short time (1–2 h) on sperm quality based on functional parameters in a population of oligo-astheno-teratozoospermia (OAT) men. The semen samples were retrieved from 50 men with OAT two times: one standard 2–7 days (long ejaculation) and short duration trimming (1–2 hours later the first ejaculation). All semen parameters as well as sperm DNA integrity were compared between groups. Results showed that mean sperm concentration (10.40 vs. 8.76), total sperm count (28.53 vs. 12.24) and mean semen volume (2.69 vs. 1.40) were higher in the first ejaculation (2–7 days of abstinence), while progressive motility (20.52 vs. 13.32), non-progressive motility (53.46 vs. 48.86), morphology (2.46 vs. 1.46) and viability (83.90 vs. 77.96) were significantly higher in the second ejaculation (P < 0.05). The second sample also showed lower immotile (26.82 vs. 38.02) and DNA fragmentation (19.5 vs. 26.96) (P < 0.05). Taking all data into account, an additional short abstinence period (AP) may be a simple and helpful strategy to obtain better sperm quality in couples with male infertility causes, especially in OAT patients. The recommended current guidelines regarding the AP may need to be revisited in severe male factors.

Dry incubators prevent bacterial growth and allow time-lapse imaging. However, they cause more significant osmotic pressure changes in the culture medium than in humidified incubators. Few reports have explored the effect of osmotic pressure on human embryos cultured under different conditions. This study examined how changes in osmotic pressure affect human embryos in a dry incubator. The study incubated embryos in culture mediums covered with mineral oil of varying viscosities and quantities. The osmotic pressure of the culture medium was measured daily for six-day period (Day 0–6) in four experimental groups established by varying the viscosity and volume of mineral oil: low viscosity (Light), 3.0 ml or 4.5 ml, and high viscosity (Heavy), 3.0 ml or 4.5 ml of mineral oil. The Light 3.0 ml and Heavy 4.5 ml groups, showing the greatest difference in the osmotic pressure, were used to culture of human embryos. After six days of incubation, the osmotic pressure increased the most in Light 3.0 ml group. Heavy 4.5 ml group had the smallest change. However, no significant differences were noted in the formation rates of blastocysts, good-quality blastocysts, or cell count between the two groups. The study suggests that even when the culture medium is covered with heavy mineral oil in a dry incubator, osmotic pressure increases after six days but does not significantly affect the formation of blastocysts. These findings provide valuable insights into the effects of varying osmotic pressure on embryonic development and may help in optimizing conditions for in vitro fertilization.

In Western diets, high consumption of meat and dairy products, known to be rich in branched-chain amino acids (BCAAs), including leucine (Leu), isoleucine (Ile), and valine (Val), as well as BCAAs supplementation itself, may have unforeseen consequences on sperm quality. In addition, bodybuilders are increasingly resorting to BCAA supplementation to build-up their muscle mass. This study aimed to assess the effect of dietary BCAAs, provided alone or in combination, on semen parameters, apoptotic gene expression, and blood amino acid (AA) profiles. To address this question and determine whether these different BCAAs have a distinct impact on sperm quality and testicular homeostasis, fifty NMRI mature male mice were exposed or not to BCAAs supplementations (control diet: CTR; CTR + Leu supplementation; CTR + Ile supplementation; CTR + Val supplementation; CTR + all three BCAA supplementation). Only valine supplementation resulted in a significant decrease in sperm concentration and viability. In addition, only valine supplementation was associated with a dramatic increase in sperm immotility. The Bax/Bcl2 ratio, an indicator of apoptosis, was found to be significantly higher in the testes of BCAA-supplemented animals when compared with the other groups. Caspase3 expression was also significantly higher in the testes of BCAA-supplemented and Val-supplemented animals. There were no significant differences in plasma AA profiles between groups. Thus, amongst BCAAs, valine supplementation appears to carry the greatest effect on sperm functional parameters and testicular apoptotic status.

During embryogenesis in Danio rerio (zebrafish), the earliest morphological patterning events are dependent on the precise temporal translation and/or localization of specific maternal mRNAs/proteins. Dorsoventral patterning in particular requires the translocation of maternal factors that are present in the Balbiani Body from the vegetal region of the unfertilized egg to the future dorsal side of the embryo (Fuentes et al., 2020), leading to the localized activation of the β-catenin pathway in the cells in that region. Since zebrafish are chordates, this dorsoventral patterning then leads to the formation of neural tissue on the dorsal side of the embryo. What is not yet clear is the identity of all maternal and zygotic factors that first establish dorsoventral patterning, and which factors lead to the establishment of neural versus non-neural tissue. Taking an evolutionary approach to this question, we investigated a gene in zebrafish, zsquidlike-A (hnrnpaba), that is homologous to a key dorsoventral patterning gene in fruit flies (Drosophila melanogaster) called squid (Kelley, 1993). While dorsoventral patterning in flies and fish looks quite different both morphologically and at the molecular level, we demonstrate that not only has a key dorsoventral patterning gene in flies been conserved in fish, maternal fish zsquidlike-A protein is synthesized precisely as dorsoventral patterning is unfolding in fish embryos, and in its absence, dorsoventral patterning is severely disrupted.

Sesquizygotic twinning (SZT) is one of the rarest events that can occur in multiple pregnancies. It has only been reported twice to date. Studies indicate that sesquizygotic twins result from the splitting of a chimeric embryo at an early stage. Theories and studies suggest that SZT represents a third unique twinning mechanism that is different from dizygotic and monozygotic twinning; however, the unique features of SZT have nothing to do with the twinning mechanism. Instead, it resembles more of an atypical monozygotic twinning form according to the twinning mechanism.

The objective of this cohort study was to investigate whether embryo quality and morphokinetic behaviour differ in the four groups of low-prognosis women as stratified by the POSEIDON criteria. The study was performed in a private university-affiliated in vitro fertilization (IVF) centre, and included 3326 injected oocytes from 846 women undergoing ICSI cycles between March 2019 and April 2022. Kinetic markers from the point of insemination were recorded in the EmbryoScope incubator. Generalized mixed models followed by Bonferroni post hoc were used to compare morphokinetics among the POSEIDON groups. Embryos derived from patients in the POSEIDON groups 2, 3 and 4 showed significantly slower divisions compared to those from POSEIDON 1 group. The KIDScore rank was significantly lower for embryos deriving from POSEIDON groups 2, 3 and 4 (2: 4.4 ± 0.7 vs. 3: 4.2 ± 0.2 vs. 4: 3.0 ± 0.4) compared to those deriving from POSEIDON 1 group (4.8 ± 0.1, p < 0.001). Group POSEIDON 1 showed improved implantation (26.9% vs. 2: 22.4% vs. 3: 20.0% vs. 4: 14.0, p < 0.001) and miscarriage rates (5.6% vs. 2: 31.2% vs. 4: 50.0%, p = 0.013). Embryo quality and morphokinetic behaviour differ across the POSEIDON groups, being more favourable in POSEIDON group 1, as well as implantation and miscarriage rates. Embryo development was more favourable in POSEIDON group 1 (young age and adequate ovarian reserve), suggesting that oocyte quality is determinant of embryo developmental potential. These findings show the reasonability of classifying POR by the POSEIDON criteria and provide information for counselling of POR regarding their possible prognosis.