Identification of genes associated with sperm storage capacity in hens at different times after insemination by RNA-seq and Ribo-seq.

BACKGROUND: Sperm storage capacity (SSC) determines the duration of fertility in hens and is an important reproduction trait that cannot be ignored in production. Currently, the genetic mechanism of SSC is still unclear in hens. Therefore, to explore the genetic basis of SSC, we analyzed the uterus-vagina junction (UVJ) of hens with different SSC at different times after insemination by RNA-seq and Ribo-seq. RESULTS: Our results showed that 589, 596, and 527 differentially expressed genes (DEGs), 730, 783, and 324 differentially translated genes (DTGs), and 804, 625, and 467 differential translation efficiency genes (DTEGs) were detected on the 5th, 10th, and 15th days after insemination, respectively. In transcription levels, we found that the differences of SSC at different times after insemination were mainly reflected in the transmission of information between cells, the composition of intercellular adhesion complexes, the regulation of ion channels, the regulation of cellular physiological activities, the composition of cells, and the composition of cell membranes. In translation efficiency (TE) levels, the differences of SSC were mainly related to the physiological and metabolic activities in the cell, the composition of the organelle membrane, the physiological activities of oxidation, cell components, and cell growth processes. According to pathway analysis, SSC was related to neuroactive ligand-receptor interaction, histidine metabolism, and PPAR signaling pathway at the transcriptional level and glutathione metabolism, oxidative phosphorylation, calcium signaling pathway, cell adhesion molecules, galactose metabolism, and Wnt signaling pathway at the TE level. We screened candidate genes affecting SSC at transcriptional levels (COL4A4, MUC6, MCHR2, TACR1, AVPR1A, COL1A1, HK2, RB1, VIPR2, HMGCS2) and TE levels(COL4A4, MUC6, CYCS, NDUFA13, CYTB, RRM2, CAMK4, HRH2, LCT, GCK, GALT). Among them, COL4A4 and MUC6 were the key candidate genes differing in transcription, translation, and translation efficiency. CONCLUSIONS: Our study used the combined analysis of RNA-seq and Ribo-seq for the first time to investigate the SSC and reveal the physiological processes associated with SSC. The key candidate genes affecting SSC were screened, and the theoretical basis was provided for the analysis of the molecular regulation mechanism of SSC.

Sperm epigenetics and male infertility: unraveling the molecular puzzle.

BACKGROUND: The prevalence of infertility among couples is estimated to range from 8 to 12%. A paradigm shift has occurred in understanding of infertility, challenging the notion that it predominantly affects women. It is now acknowledged that a significant proportion, if not the majority, of infertility cases can be attributed to male-related factors. Various elements contribute to male reproductive impairments, including aberrant sperm production caused by pituitary malfunction, testicular malignancies, aplastic germ cells, varicocele, and environmental factors. MAIN BODY: The epigenetic profile of mammalian sperm is distinctive and specialized. Various epigenetic factors regulate genes across different levels in sperm, thereby affecting its function. Changes in sperm epigenetics, potentially influenced by factors such as environmental exposures, could contribute to the development of male infertility. CONCLUSION: In conclusion, this review investigates the latest studies pertaining to the mechanisms of epigenetic changes that occur in sperm cells and their association with male reproductive issues.

Moderate concentration of Lactobacillus metabolites does not adversely affect mouse sperm.

Many couples in contemporary societies suffer from infertility of unexplained origins (idiopathic). A promising treatment strategy within this context involves the administration to women of preparations containing lactic acid bacteria (Lactobacillus) and/or their metabolites. Recent investigations underscore the role of lactobacilli in sustaining female fertility and enhancing the effectiveness of assisted reproductive techniques. There have also been reports describing the effect of lactobacilli on sperm functions, but our knowledge in this domain remains uncertain. In this study, the effect of supernatant from Lactobacillus rhamnosus culture on mouse sperm viability and motility was tested. The protective properties of lactobacilli metabolites against hydrogen peroxide-induced DNA damage were also verified. It was shown that the metabolites have no effect on viability, motility, and genome integrity of spermatozoa, but in excessive concentrations they become toxic. The obtained results imply that probiotic and/or postbiotic preparations taken by women should not adversely affect the sperm of their partners, provided the dose is correctly selected.

Sex-specific transgenerational effects on murine thyroid gland imposed by ancestral exposure to neonicotinoid thiacloprid.

Neonicotinoids, a relatively new widely used class of insecticide is used in agriculture to control insect populations. We examined the capacity of ancestral exposure to the neonicotinoid thiacloprid (thia) to induce transgenerational effects on thyroid tissue. Pregnant outbred Swiss female mice were exposed to thia at embryonic days E6.5 to E15.5 using 0, 0.6, and 6 mg/kg/day doses. Thyroid paraffin sections were prepared for morphology analysis. We apply ELISA method to measure T4 and TSH levels, RT-qPCR for gene expression analysis, ChIP-qPCR techniques for sperm histone H3K4me3 analysis, and immunofluorescence microscopy and western blots for protein detection. We observed an alteration in the morphology of thyroids in both males and females in the F3 generation. We observed an increase in T4 hormone in F1 females and a significant T4 level decrease in F3 males. T4 changes in F1 females were associated with a TSH increase. We found that the amount of Iodothyronine Deiodinase 1 (DIO1) (an enzyme converting T4 to T3) was decreased in both F1 and F3 generations in female thyroids. GNAS protein which is important for thyroid function has increased in female thyroids. Gene expression analysis showed that the expression of genes encoding thyroid gland development, chromatin, biosynthesis and transport factors were affected in the thyroid gland in both sexes in F1 and F3. The analysis of sperm histone H3K4me3 showed that H3K4me3 occupancy at the Dio1 locus has decreased while Thyroglobulin (Tg) and Matrix Metallopeptidase 2 (Mmp2) genes have increased H3K4me3 occupancy in the sperm of F3 mice. Besides, DNA methylation analysis of our previously published datasets showed that, in the sperm of F1 and F3 thia-derived mice, several genes related to thyroid function show consistent alterations. Our data suggest that ancestral exposure to thiacloprid affects thyroid function not only in exposed but also in indirectly exposed F3 generation.

Genotyping of rams based on melatonin receptor 1A gene polymorphisms: a tool in sire selection?

Context Several polymorphisms in the melatonin receptor 1A gene (MTNR1A ) have been related to reproductive performance in ovine. Aims To investigate the effect of the Rsa I and Mnl I polymorphisms on ram seminal quality. Methods Eighteen Rasa Aragonesa rams were genotyped for the Rsa I (C/C, C/T, T/T) and Mnl I (G/G, G/A, A/A) allelic variants of the MTNR1A gene. Individual ejaculates were analysed once a month throughout the whole year. Sperm motility, morphology, membrane integrity, levels of reactive oxygen species (ROS), phosphatidylserine (PS) inversion, DNA fragmentation and capacitation status were assessed. The effect of the season and polymorphisms on seminal quality was evaluated by mixed ANOVA. Key results Both polymorphisms had an effect on membrane integrity and viable spermatozoa with low levels of ROS and without PS translocation, and Rsa I also on motile and DNA-intact spermatozoa. An interaction between both polymorphisms was found, pointing to a negative effect on seminal quality of carrying the T or A allele in homozygosity. Differences were higher in the reproductive than in the non-reproductive season. Conclusions Mutations substituting C by T and G by A at Rsa I and Mnl I polymorphic sites, respectively, in the MTNR1A gene in rams could decrease the seminal quality. Implications Genotyping of rams based on melatonin receptor 1A could be a powerful tool in sire selection.

Single-cell and spatial transcriptomic investigation reveals the spatiotemporal specificity of the beta-defensin gene family during mouse sperm maturation.

Low sperm motility is a significant contributor to male infertility. beta-defensins have been implicated in host defence and the acquisition of sperm motility; however, the regulatory mechanisms governing their gene expression patterns and functions remain poorly understood. In this study, we performed single-cell RNA and spatial transcriptome sequencing to investigate the cellular composition of testicular and epididymal tissues and examined their gene expression characteristics. In the epididymis, we found that epididymal epithelial cells display a region specificity of gene expression in different epididymal segments, including the beta-defensin family genes. In particular, Defb15, Defb18, Defb20, Defb25 and Defb48 are specific to the caput; Defb22, Defb23 and Defb26 to the corpus; Defb2 and Defb9 to the cauda of the epididymis. To confirm this, we performed mRNA fluorescence in situ hybridisation (FISH) targeting certain exon region of beta-defensin genes, and found some of their expression matched the sequencing results and displayed a close connection with epididimosome marker gene Cd63. In addition, we paid attention to the Sertoli cells and Leydig cells in the testis, along with fibroblasts and smooth muscle cells in the epididymis, by demonstrating their gene expression profile and spatial information. Our study provides a single-cell and spatial landscape for analysing the gene expression characteristics of testicular and epididymal environments and has important implications for the study of spermatogenesis and sperm maturation.

The mechanoreceptor Piezo is required for spermatogenesis in Bombyx mori.

BACKGROUND: The animal sperm shows high diversity in morphology, components, and motility. In the lepidopteran model insect, the silkworm Bombyx mori, two types of sperm, including nucleate fertile eupyrene sperm and anucleate unfertile apyrene sperm, are generated. Apyrene sperm assists fertilization by facilitating the migration of eupyrene spermatozoa from the bursa copulatrix to the spermatheca. During spermatogenesis, eupyrene sperm bundles extrude the cytoplasm by peristaltic squeezing, while the nuclei of the apyrene sperm bundles are discarded with the same process, forming matured sperm. RESULTS: In this study, we describe that a mechanoreceptor BmPiezo, the sole Piezo ortholog in B. mori, plays key roles in larval feeding behavior and, more importantly, is essential for eupyrene spermatogenesis and male fertility. CRISPR/Cas9-mediated loss of BmPiezo function decreases larval appetite and subsequent body size and weight. Immunofluorescence analyses reveal that BmPiezo is intensely localized in the inflatable point of eupyrene sperm bundle induced by peristaltic squeezing. BmPiezo is also enriched in the middle region of apyrene sperm bundle before peristaltic squeezing. Cytological analyses of dimorphic sperm reveal developmental arrest of eupyrene sperm bundles in BmPiezo mutants, while the apyrene spermatogenesis is not affected. RNA-seq analysis and q-RT-PCR analyses demonstrate that eupyrene spermatogenic arrest is associated with the dysregulation of the actin cytoskeleton. Moreover, we show that the deformed eupyrene sperm bundles fail to migrate from the testes, resulting in male infertility due to the absence of eupyrene sperm in the bursa copulatrix and spermatheca. CONCLUSIONS: In conclusion, our studies thus uncover a new role for Piezo in regulating spermatogenesis and male fertility in insects.

Reactive oxygen species and their role in the andrological factor of couple fertility.

Reactive oxygen species play a significant role in male fertility and infertility. They are essential for physiological processes, but when their concentration becomes excessive, they can be a cause of various sperm pathologies. Seminal leukocytes and pathologically abnormal sperm are the primary sources of oxygen radicals in ejaculate. They negatively affect sperm quality, including DNA fragmentation and sperm motility impairment. Addressing increased concentrations of reactive oxygen species involves various appropriate lifestyle modifications and measures, including the use of antioxidants, treatment of urogenital infections, management of varicocele, weight reduction, and others. In many cases, these interventions can lead to adjustments in the condition and improvement in sperm quality. Such improvements can subsequently lead to enhanced outcomes in assisted reproduction or even an increased likelihood of natural conception. In some instances, the need for donor sperm may be eliminated. However, a key factor is adhering to a sufficiently prolonged treatment, which requires patience on the part of both, the physician and the patient.

Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

Trim66's paternal deficiency causes intrauterine overgrowth.

The tripartite motif-containing protein 66 (TRIM66, also known as TIF1-delta) is a PHD-Bromo-containing protein primarily expressed in post-meiotic male germ cells known as spermatids. Biophysical assays showed that the TRIM66 PHD-Bromodomain binds to H3 N-terminus only when lysine 4 is unmethylated. We addressed TRIM66's role in reproduction by loss-of-function genetics in the mouse. Males homozygous for Trim66-null mutations produced functional spermatozoa. Round spermatids lacking TRIM66 up-regulated a network of genes involved in histone acetylation and H3K4 methylation. Profiling of H3K4me3 patterns in the sperm produced by the Trim66-null mutant showed minor alterations below statistical significance. Unexpectedly, Trim66-null males, but not females, sired pups overweight at birth, hence revealing that Trim66 mutations cause a paternal effect phenotype.

Uncommon opsin's retinal isomer is involved in mammalian sperm thermotaxis.

In recent years it became apparent that, in mammals, rhodopsin and other opsins, known to act as photosensors in the visual system, are also present in spermatozoa, where they function as highly sensitive thermosensors for thermotaxis. The intriguing question how a well-conserved protein functions as a photosensor in one type of cells and as a thermosensor in another type of cells is unresolved. Since the moiety that confers photosensitivity on opsins is the chromophore retinal, we examined whether retinal is substituted in spermatozoa with a thermosensitive molecule. We found by both functional assays and mass spectrometry that retinal is present in spermatozoa and required for thermotaxis. Thus, starvation of mice for vitamin A (a precursor of retinal) resulted in loss of sperm thermotaxis, without affecting motility and the physiological state of the spermatozoa. Thermotaxis was restored after replenishment of vitamin A. Using reversed-phase ultra-performance liquid chromatography mass spectrometry, we detected the presence of retinal in extracts of mouse and human spermatozoa. By employing UltraPerformance convergence chromatography, we identified a unique retinal isomer in the sperm extracts-tri-cis retinal, different from the photosensitive 11-cis isomer in the visual system. The facts (a) that opsins are thermosensors for sperm thermotaxis, (b) that retinal is essential for thermotaxis, and (c) that tri-cis retinal isomer uniquely resides in spermatozoa and is relatively thermally unstable, suggest that tri-cis retinal is involved in the thermosensing activity of spermatozoa.

Dataset of seminal plasma proteome of Nellore bulls with high and low percentage of abnormalities sperm.

OBJECTIVES: Bovine seminal plasma proteins perform several functions related to sperm function. Changes in the expression pattern or abundance of seminal proteins are related to changes in the fertilizing capacity of bulls. Considering the role of seminal plasma proteins in sperm function and animal reproduction, we investigated changes in the protein abundance profile in response to sperm morphological changes using a proteomic approach. DATADESCRIPTION: In our present investigation, we employed liquid chromatography coupled with mass spectrometry to elucidate the proteomic composition of seminal plasma obtained from Nellore bulls exhibiting varying percentages of sperm abnormalities. Following semen collection, seminal plasma was promptly isolated from sperm, and proteins were subsequently precipitated, enzymatically digested using porcine trypsin, and subjected to analysis utilizing the Acquity nano UHPLC System in conjunction with a mass spectrometer. This dataset encompasses a total of 297 proteins, marking the inaugural instance in which a comparative profile of seminal plasma proteins in young Nellore bulls, categorized by their sperm abnormality percentages, has been delineated using LC-MS/MS. The comprehensive nature of this dataset contributes pivotal proteomic insights, representing a noteworthy advancement in our understanding of the reproductive biology of the Nellore breed.

Retinoic acid mitigates the NSC319726-induced spermatogenesis dysfunction through cuproptosis-independent mechanisms.

Copper ionophore NSC319726 has attracted researchers' attention in treating diseases, particularly cancers. However, its potential effects on male reproduction during medication are unclear. This study aimed to determine whether NSC319726 exposure affected the male reproductive system. The reproductive toxicity of NSC319726 was evaluated in male mice following a continuous exposure period of 5 weeks. The result showed that NSC319726 exposure caused testis index reduction, spermatogenesis dysfunction, and architectural damage in the testis and epididymis. The exposure interfered with spermatogonia proliferation, meiosis initiation, sperm count, and sperm morphology. The exposure also disturbed androgen synthesis and blood testis barrier integrity. NSC319726 treatment could elevate the copper ions in the testis to induce cuproptosis in the testis. Copper chelator rescued the elevated copper ions in the testis and partly restored the spermatogenesis dysfunction caused by NSC319726. NSC319726 treatment also decreased the level of retinol dehydrogenase 10 (RDH10), thereby inhibiting the conversion of retinol to retinoic acid, causing the inability to initiate meiosis. Retinoic acid treatment could rescue the meiotic initiation and spermatogenesis while not affecting the intracellular copper ion levels. The study provided an insight into the bio-safety of NSC319726. Retinoic acid could be a potential therapy for spermatogenesis impairment in patients undergoing treatment with NSC319726.

Aquaporin expression in cryopreserved human sperm: exploring the capabilities of natural deep eutectic solvents (NADEs).

BACKGROUND: Aquaporins (AQPs) are essential proteins that facilitate the rapid movement of water and cryoprotective agents (CPAs) during the cryopreservation process, and ensure the cryo-tolerance of sperm cells. OBJECTIVE: This study evaluated the preservation of aquaporin levels in human sperm after undergoing freezing using natural deep eutectic solvents (NADES) as CPAs for cryoprotection. MATERIALS AND METHODS: From June 2021 to October 2022, 35 semen samples with normal sperm parameters were acquired from the Mehr Infertility Treatment Institute in Rasht, Iran. The samples were divided into several groups for analysis: control group (not frozen), group frozen with SpermFreeze Solution, and groups frozen with different NADESs, including ChS, ChX, ChU, ChG, GlyP, and EtP. After thawing, various aspects for each group were assessed, including the integrity and condensation of sperm chromatin, viability, motility, integrity of acrosome, and the expression of AQP1, AQP3, AQP7, AQP8, and AQP9 genes. RESULTS: The analysis of gene expression revealed that freezing with ChS and GlyP preserved the expression of the AQP1 and AQP3 genes compared to the control group. Regarding AQP7 and AQP8, significant differences were not observed in expression levels between certain NADES groups (e.g., ChS, ChU, and GlyP) and the control group. Additionally, samples frozen with specific NADESs, such as ChS, ChG, EtP, and GlyP, exhibited preserved levels of AQP9 expression when compared to the control group. CONCLUSION: These findings emphasize the importance of NADES in preserving the expression of aquaporins in cryopreserved human sperm and their important fertility parameters. Doi.org/10.54680/fr24310110512.

Evaluation of the aquaporin molecules characterization in the sperm cells of men from different aged.

Background/aim: Male infertility rises for many reasons, along with age; therefore, we aimed to research the characterization of aquaporin-3, 7, and 8 in human sperm belonging to different age groups. Material and methods: This study was conducted on sperm samples of men aged over 18 years. A total of 60 men were included in the study and divided into three age groups: group 1, age 18-25 years (n = 20); group 2, age 26-35 years (n = 20); and group 3, age ≥35 years (n = 20). Sperm ejaculates obtained from each participant were used for spermiogram tests, Kruger strict morphology analysis, and immunohistochemistry. Results: We observed no statistically significant differences in terms of macroscopic and microscopic sperm testing. The immunostaining score of aquaporin-3 was the lowest in group 1 and increased in group 3 and group 2, respectively (p < 0.05). Aquaporin-8 immunostaining only increased in group 2 (p < 0.05). Aquaporin-7 immunostaining scores were not different between the groups (p > 0.05). When the immunostaining scores of aquaporin molecules were compared with each other, aquaporin-7 was significantly increased compared with the others (p < 0.05). Conclusion: According to the results, it can be stated that aquaporin-3 and aquaporin-8 molecules were more expressed at age 26 to 35 years, and aquaporin-7 was densely expressed from age 18 to 25 years. If the characterization of these molecules is adversely affected, male infertility may eventually emerge. We recommend further advanced-level studies on this subject.

Supplementation of sperm cryopreservation media with H2S donors enhances sperm quality, reduces oxidative stress, and improves in vitro fertilization outcomes.

Cryopreservation of sperm can cause oxidative stress and damage, leading to decreased different functional parameters and fertilization potential. In this study, we evaluated two types of H2S donors: NaHS, a fast-releasing donor, and GYY4137, a slow-releasing donor during cryopreservation of goat sperm. Initially, we determined that 1.5 and 3 µM NaHS, and 15 and 30 µM GYY4137 are optimal concentrations that improved different sperm functional parameters including motility, viability, membrane integrity, lipid peroxidation, and ROS production during incubation at 38.5 °C for 90 min. We subsequently evaluated the impact of the optimal concentration of NaHS and GYY4137 supplementation on various functional parameters following thawing during cryopreservation. Our data revealed that supplementation of extender improved different parameters including post-thaw sperm motility, viability, membrane integrity, and reduced DNA damage compared to the frozen-thawed control group. The supplementation also restored the redox state, decreased lipid peroxidation, and improved mitochondrial membrane potential in the thawed sperm. Finally, we found that supplementation of the extender with NaHS and GYY4137 enhanced IVF outcomes in terms of blastocyst rate and quality of blastocysts. Our results suggest that both donors can be applied for cryopreservation as antioxidants to improve sperm quality and IVF outcomes of frozen-thawed goat sperm.

Type 1 diabetes impairs the activity of rat testicular somatic and germ cells through NRF2/NLRP3 pathway-mediated oxidative stress.

Background: It is well known that metabolic disorders, including type 1 diabetes (T1D), are often associated with reduced male fertility, mainly increasing oxidative stress and impairing the hypothalamus-pituitary-testis (HPT) axis, with consequently altered spermatogenesis and reduced sperm parameters. Herein, using a rat model of T1D obtained by treatment with streptozotocin (STZ), we analyzed several parameters of testicular activity. Methods: A total of 10 adult male Wistar rats were divided into two groups of five: control and T1D, obtained with a single intraperitoneal injection of STZ. After 3 months, the rats were anesthetized and sacrificed; one testis was stored at -80°C for biochemical analysis, and the other was fixed for histological and immunofluorescence analysis. Results: The data confirmed that T1D induced oxidative stress and, consequently, alterations in both testicular somatic and germ cells. This aspect was highlighted by enhanced apoptosis, altered steroidogenesis and Leydig cell maturity, and impaired spermatogenesis. In addition, the blood-testis barrier integrity was compromised, as shown by the reduced levels of structural proteins (N-cadherin, ZO-1, occludin, connexin 43, and VANGL2) and the phosphorylation status of regulative kinases (Src and FAK). Mechanistically, the dysregulation of the SIRT1/NRF2/MAPKs signaling pathways was proven, particularly the reduced nuclear translocation of NRF2, affecting its ability to induce the transcription of genes encoding for antioxidant enzymes. Finally, the stimulation of testicular inflammation and pyroptosis was also confirmed, as highlighted by the increased levels of some markers, such as NF-κB and NLRP3. Conclusion: The combined data allowed us to confirm that T1D has detrimental effects on rat testicular activity. Moreover, a better comprehension of the molecular mechanisms underlying the association between metabolic disorders and male fertility could help to identify novel targets to prevent and treat fertility disorders related to T1D.

Unlocking Genetic Mysteries during the Epic Sperm Journey toward Fertilization: Further Expanding Cre Mouse Lines.

The spatiotemporal expression patterns of genes are crucial for maintaining normal physiological functions in animals. Conditional gene knockout using the cyclization recombination enzyme (Cre)/locus of crossover of P1 (Cre/LoxP) strategy has been extensively employed for functional assays at specific tissue or developmental stages. This approach aids in uncovering the associations between phenotypes and gene regulation while minimizing interference among distinct tissues. Various Cre-engineered mouse models have been utilized in the male reproductive system, including Dppa3-MERCre for primordial germ cells, Ddx4-Cre and Stra8-Cre for spermatogonia, Prm1-Cre and Acrv1-iCre for haploid spermatids, Cyp17a1-iCre for the Leydig cell, Sox9-Cre for the Sertoli cell, and Lcn5/8/9-Cre for differentiated segments of the epididymis. Notably, the specificity and functioning stage of Cre recombinases vary, and the efficiency of recombination driven by Cre depends on endogenous promoters with different sequences as well as the constructed Cre vectors, even when controlled by an identical promoter. Cre mouse models generated via traditional recombination or CRISPR/Cas9 also exhibit distinct knockout properties. This review focuses on Cre-engineered mouse models applied to the male reproductive system, including Cre-targeting strategies, mouse model screening, and practical challenges encountered, particularly with novel mouse strains over the past decade. It aims to provide valuable references for studies conducted on the male reproductive system.

Cytosolic and Acrosomal pH Regulation in Mammalian Sperm.

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.

Proteomic diversification of spermatostyles among six species of whirligig beetles.

Seminal fluid protein composition is complex and commonly assumed to be rapidly divergent due to functional interactions with both sperm and the female reproductive tract (FRT), both of which evolve rapidly. In addition to sperm, seminal fluid may contain structures, such as mating plugs and spermatophores. Here, we investigate the evolutionary diversification of a lesser-known ejaculate structure: the spermatostyle, which has independently arisen in several families of beetles and true bugs. We characterized the spermatostyle proteome, in addition to spermatostyle and FRT morphology, in six species of whirligig beetles (family Gyrinidae). Spermatostyles were enriched for proteolytic enzymes, and assays confirmed they possess proteolytic activity. Sperm-leucylaminopeptidases (S-LAPs) were particularly abundant, and their localization to spermatostyles was confirmed by immunohistochemistry. Although there was evidence for functional conservation of spermatostyle proteomes across species, phylogenetic regressions suggest evolutionary covariation between protein composition and the morphology of both spermatostyles and FRTs. We postulate that S-LAPs (and other proteases) have evolved a novel structural role in spermatostyles and discuss spermatostyles as adaptations for delivering male-derived materials to females.