Carine Dahl Corcini

While lipophilic antioxidants such as flavonols may improve the preservation capacity of sperm at 17°C, their low stability and bioavailability can reduce their biological effectiveness. Encapsulation strategies have been proposed to improve the delivery and cellular availability of hydrophobic antioxidants. Herein, we investigated whether delivering one of these flavonols (kaempferol) in nanoliposomes can improve the preservation capacity of pig semen at 17°C. Twelve semen samples were supplemented with kaempferol‐loaded nanoliposomes (KNL) or free kaempferol (5 μM), and stored at 17°C for 48 h. Sperm motility was assessed using a computer‐assisted sperm analysis system, and functional parameters were evaluated by flow cytometry. After 48 h, sperm stored with KNL showed lower intracellular reactive oxygen species and superoxide levels than those stored with free kaempferol. In addition, the integrity of the plasma membrane and acrosome was better maintained in samples containing KNL. Conversely, mitochondrial membrane potential and most kinematic parameters in sperm stored with KNL did not differ from those supplemented with free kaempferol, yet curvilinear velocity was higher in the former than in the latter. These findings indicate that nanoliposomal delivery enhances the biological effectiveness of 5 μM kaempferol, improving its antioxidant and protective effects on pig sperm. Further research is warranted to improve sperm preservation capacity by delivering lipophilic antioxidants via nanoliposomes.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

Graphical Cyclophosphamide administration often causes infertility. Ovarian puncture (OP), a component of in vitro fertilization, is performed to obtain oocytes. Our previous study showed that OP stimulated follicular activation in mice. However, its effects on folliculogenesis in cyclophosphamide-treated animals remain unclear. This study aimed to evaluate the impact of OP on follicular growth and gene expression. Female C57BL/06J mice (n = 52) were divided into three groups (control, cyclo, and cyclo + Pc). Cyclophosphamide was administered in four doses to the cyclo and cyclo + Pc groups (150 mg/kg), and OP was performed three days after the last dose and 96 h before euthanasia in the cyclo + Pc group. There was no difference in body weight; however, ovarian weight was higher in cyclo + Pc compared with cyclo. The number of all follicle classes was similar between cyclo + Pc and cyclo. Caspase-3-positive granulosa cells (%) in antral follicles were increased in cyclo compared with control and reduced in cyclo + Pc compared with control. Amh expression was increased in the cyclo + Pc group compared with the cyclo group. Pten expression was reduced in cyclo + Pc, whereas Kitl expression was increased. Bax expression was reduced, and there was no change in Casp3 expression due to OP. Moreover, collagen deposition was increased in cyclo + Pc compared with the control and cyclo groups. However, the treatments did not change the ovulation response, oocytes, or follicular morphometrics. Our experimental study demonstrates, for the first time, that OP is a safe procedure that can be performed during cyclophosphamide treatment in mice. Lay summary: Infertility is a concern for cancer patients who wish to conceive after completing chemotherapy treatment. Puncturing the ovary is part of the IVF procedure, aimed at collecting eggs to fertilize and transferring them to the uterus. We decided to study whether the inflammation and tissue damage caused by puncturing the ovaries could stimulate the collection of healthy eggs in patients who have gone through chemotherapy. To this end, we used female mice that were treated with a frequently used treatment for breast cancer, along with ovarian puncture. We discovered the alterations did not affect follicle growth or egg retrieval. With these findings, we demonstrated, for the first time, the ability of ovarian puncture to bring about healthy egg retrieval, improving quality of life after cancer.

Lectins are carbohydrate‐binding proteins that recognize specific glycan patterns on cell surfaces and have been increasingly explored as analytical tools in reproductive biology. This study characterized the interaction of a lectin isolated from Abelmoschus esculentus seeds (AEL) with ovine spermatozoa and evaluated its compatibility with sperm functional parameters during short‐term refrigeration. Eighteen ejaculates were diluted in a Tris–egg yolk extender supplemented with 0, 1, or 3 µg/mL AEL and stored at 5°C for 24 h. Sperm motility (CASA) and flow cytometry were used to assess plasma membrane integrity and fluidity, mitochondrial membrane potential, and reactive oxygen species production. Confocal microscopy was performed to determine the localization pattern of AEL binding. AEL labeling was predominantly restricted to the sperm midpiece region. Supplementation with AEL at the tested concentrations did not affect motility patterns, membrane integrity, mitochondrial membrane potential, or ROS levels after 24 h of storage ( p > 0.05). These findings indicate that AEL exhibits a consistent midpiece‐associated binding pattern and does not compromise sperm functional parameters under refrigerated conditions. The results support the use of AEL as a compatible probe for investigating glycan distribution in the midpiece region of ram spermatozoa.

Delivery of large molecular complexes into spermatozoa remains a challenge in reproductive biotechnology. Teleost sperm possess highly compacted chromatin and minimal cytoplasmic volume, limiting intracellular access of ribonucleoprotein assemblies. Here, we evaluated whether CRISPR‐Cas9 ribonucleoprotein (RNP) complexes can be introduced into Nile tilapia spermatozoa using capillary electroporation while preserving cellular integrity. RNP uptake was quantified via EGFP fluorescence, and sperm performance was assessed through motility, membrane integrity, mitochondrial activity, and DNA fragmentation analyses. Approximately 10%–13% of spermatozoa exhibited detectable EGFP signal following electroporation. Increased pulse numbers reduced motility and mitochondrial activity, whereas membrane integrity was preserved and no increase in DNA fragmentation was detected. A single pulse (1250 V, 40 ms) provided the best balance between RNP uptake and functional preservation. These findings support capillary electroporation as a minimally disruptive strategy for CRISPR‐RNP delivery in teleost reproductive biology.

Interest in male fertility is relatively recent. The first records date back to 1677, when Anton Philips van Leeuwenhoek observed spermatozoa for the first time under his microscope, referring to t...

Isoflurane is a widely used inhalational anesthetic. Occupational exposure to waste anesthetic gases (WAGs) raises concerns about DNA damage. Bone marrow, highly susceptible due to its proliferative activity, has been scarcely investigated in rodents after exposure to WAGs. Therefore, the present study evaluated the impact of this exposure on micronucleus (MN) frequency and cytotoxicity, under conditions that mimic work environments with WAG to which thousands of professionals worldwide are occupationally exposed. Swiss mice were randomized into control, exposed, and recovery groups. Animals were subjected to daily 5 h exposures to 50 ppm isoflurane for 30 days. Bone marrow samples were analyzed using the MN assay. Isoflurane‐exposed mice showed a significant increase in micronucleated polychromatic erythrocytes compared to controls ( p < 0.0001). The recovery group exhibited a partial reduction after 20 days without exposure, though values remained above the control. Simulated occupational isoflurane exposure induces chromosomal instability in bone marrow cells, even at recommended safety levels.

Introdução: O isoflurano, amplamente utilizado em ambientes médicos e científicos, desempenha um papel significativo na geração de gases anestésicos residuais. A exposição ocupacional a este anestésico pode resultar em graves prejuízos à saúde devido à exposição crônica, principalmente no que diz respeito ao impacto oxidativo. O fígado, sendo o principal órgão responsável pelo metabolismo deste anestésico, é um alvo chave para estes efeitos adversos. Objetivo: Avaliar os efeitos da exposição ocupacional crônica ao isoflurano sobre o estado redox hepático de camundongos e investigar se a interrupção da exposição pode restaurar o equilíbrio oxidativo. Métodos: Os ratos foram expostos a 50 ppm de isoflurano durante 5 horas por dia durante um período de 30 dias. Após a exposição, eles foram sacrificados no primeiro dia após o término da exposição (G2) ou 20 dias após a última exposição (G3) e comparados ao grupo controle (G1). Suas amostras de tecido hepático foram submetidas a análises bioquímicas, avaliando-se a concentração de espécies reativas de oxigênio e substâncias reativas ao ácido tiobarbitúrico, bem como a atividade das enzimas catalase e superóxido dismutase. Resultados: A exposição ocupacional ao isoflurano pode afetar a atividade de enzimas antioxidantes no tecido hepático. Os indivíduos expostos apresentaram redução da atividade da catalase, associada a um aumento tardio e não fisiológico da atividade da superóxido dismutase. Conclusão: O isoflurano em concentrações supostamente seguras perturba o estado redox hepático dos indivíduos expostos, e a interrupção da exposição não garante o retorno ao equilíbrio oxidativo.