Defective Spermatogenesis Research Articles

Deviations in the Z:A ratio disrupt sexual development in the eri silkmoth, Samia cynthia ricini.

Moths and butterflies (Lepidoptera) have sex chromosome systems with female heterogamety, and 2 models, W-dominance and Z-counting, have been proposed to determine sex. The W-dominant mechanism is well known in Bombyx mori. However, little is known about the Z-counting mechanism in Z0/ZZ species. We investigated whether ploidy changes affect sexual development and gene expression in the eri silkmoth, Samia cynthia ricini (2n = 27♀/28♂, Z0♀/ZZ♂). Tetraploid males (4n = 56, ZZZZ) and females (4n = 54, ZZ) were induced by heat and cold shock, and then, triploid embryos were produced by crosses between diploids and tetraploids. Two karyotypes (3n = 42, ZZZ and 3n = 41, ZZ) were identified in triploid embryos. Triploid embryos with 3 Z chromosomes showed male-specific splicing of the S. cynthia doublesex (Scdsx) gene, whereas 2-Z triploid embryos showed both male- and female-specific splicing. From larva to adult, 3-Z triploids showed a normal male phenotype, except for defects in spermatogenesis. However, abnormal gonads were observed in 2-Z triploids, which showed both male- and female-specific Scdsx transcripts not only in the gonads but also in somatic tissues. Two-Z triploids were thus obviously intersexes, suggesting that sexual development in S. c. ricini depends on the Z:A ratio and not only on the Z number. Moreover, mRNA-seq analyses in embryos showed that relative levels of gene expression are similar between samples with different doses of Z chromosomes and autosome sets. Our results provide the first evidence that ploidy changes disrupt sexual development but have no effect on the general mode of dosage compensation in Lepidoptera.

Cytochrome c1-like is required for mitochondrial morphogenesis and individualization during spermatogenesis in Drosophila melanogaster.

The Drosophila testis is an excellent system for studying the process from germ stem cells to motile sperm, including the proliferation of male germ cells, meiosis of primary spermatocytes, mitochondrial morphogenesis, and spermatid individualization. We previously demonstrated that ocnus (ocn) plays an essential role in male germ cell development. Among those genes and proteins whose expression levels were changed as a result of ocn knockdown, cytochrome c1-like (cyt-c1L) was downregulated significantly. Here, we show that cyt-c1L is highly expressed in the testis of D. melanogaster. Knockdown or mutation of cyt-c1L in early germ cells of flies resulted in male sterility. Immunofluorescence staining showed that cyt-c1L knockdown testes had no defects in early spermatogenesis; however, in late stages, in contrast to many individualization complexes (ICs) composed of F-actin cones that appeared at different positions in control testes, no actin cones or ICs were observed in cyt-c1L knockdown testes. Furthermore, no mature sperm were found in the seminal vesicle of cyt-c1L knockdown testes whereas the control seminal vesicle was full of mature sperm with needle-like nuclei. cyt-c1L knockdown also caused abnormal mitochondrial morphogenesis during spermatid elongation. Excessive apoptotic signals accumulated in the base of cyt-c1L knockdown fly testes. These results suggest that cyt-c1L may play an important role in spermatogenesis by affecting the mitochondrial morphogenesis and individualization of sperm in D. melanogaster.

The Prmt5-Vasa module is essential for spermatogenesis in Bombyx mori.

In lepidopteran insects, dichotomous spermatogenesis produces eupyrene spermatozoa, which are nucleated, and apyrene spermatozoa, which are anucleated. Both sperm morphs are essential for fertilization, as eupyrene sperm fertilize the egg, and apyrene sperm is necessary for the migration of eupyrene sperm. In Drosophila, Prmt5 acts as a type II arginine methyltransferase that catalyzes the symmetrical dimethylation of arginine residues in the RNA helicase Vasa. Prmt5 is critical for the regulation of spermatogenesis, but Vasa is not. To date, functional genetic studies of spermatogenesis in the lepidopteran model Bombyx mori has been limited. In this study, we engineered mutations in BmPrmt5 and BmVasa through CRISPR/Cas9-based gene editing. Both BmPrmt5 and BmVasa loss-of-function mutants had similar male and female sterility phenotypes. Through immunofluorescence staining analysis, we found that the morphs of sperm from both BmPrmt5 and BmVasa mutants have severe defects, indicating essential roles for both BmPrmt5 and BmVasa in the regulation of spermatogenesis. Mass spectrometry results identified that R35, R54, and R56 of BmVasa were dimethylated in WT while unmethylated in BmPrmt5 mutants. RNA-seq analyses indicate that the defects in spermatogenesis in mutants resulted from reduced expression of the spermatogenesis-related genes, including BmSxl, implying that BmSxl acts downstream of BmPrmt5 and BmVasa to regulate apyrene sperm development. These findings indicate that BmPrmt5 and BmVasa constitute an integral regulatory module essential for spermatogenesis in B. mori.

DMRT1, RBMY, and AZFb genes polymorphism and expression role in azoospermia susceptibility

Male infertility can occur due to spermatogenesis defects. The most common causes of male infertility are azoospermia and oligospermia, which have several underlying factors, one of which is genetic. This study aimed to investigate the association of azoospermia with the DMRT1 and RBMY1A1 genes polymorphisms and AZFb region microdeletions in Iranian men. Moreover, these genes expression were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). A total of 100 Iranian men with azoospermia, oligozoospermia, or severe oligozoospermia and 100 fertile controls were included in this case-control study. A total of 200 subjects were genotyped for DMRT1 rs755383 and RBMY1A1 rs1481942953 polymorphisms using Tetra-ARMS PCR. The presence of two sequence-tagged sites (STS) markers from the Y chromosome AZFb region was also investigated by multiplex PCR. RT-PCR was used to analyze the expression in the testis tissue of azoospermia patients. With a P-value of 0.038, rs755383 in the DMRT1 gene was associated with an increased risk of azoospermia. However, no significant difference was found in genotype distribution in the RBMY1A1 (rs1481942953) gene polymorphism. Four patients showed Y chromosome microdeletions with sY127 and sY134 markers in the AZFb region. Infertile males' cDNA analysis revealed low expression levels for DMRT1 and PRY (one of the main genes in the AZFb region) with a p-value<0.0001. In contrast, RBMY1A1 expression level did not differ between patients and control groups with a p-value of 0.112. A receiver operating characteristic (ROC) curve analysis was carried out to detect genes with biomarker potential. With AUCs of 83% and 77%, DMRT1 and PRY had diagnostic marker potential in azoospermia detection.

Cutaneous Melanoma and Hormones: Focus on Sex Differences and the Testis.

Cutaneous melanoma, the most aggressive type of skin cancer, remains one the most represented forms of cancer in the United States and European countries, representing, in Australia, the primary cause of cancer-related deaths. Recently, many studies have shown that sex disparities previously observed in most cancers are particularly accentuated in melanoma, where male sex is consistently associated with an increased risk of disease progression and a higher mortality rate. The causes of these sex differences rely on biological mechanisms related to sex hormones, immune homeostasis and oxidative processes. The development of newer therapies, such as immune checkpoint inhibitors (ICIs) (i.e., anti-PD-1 and anti-CTLA-4 monoclonal antibodies) has dramatically changed the treatment landscape of metastatic melanoma patients, though ICIs can interfere with the immune response and lead to inflammatory immune-related adverse events (irAEs). Recently, some studies have shown a potential adverse influence of this immunotherapy treatment also on male fertility and testicular function. However, while many anticancer drugs are known to cause defects in spermatogenesis, the effects of ICIs therapy remain largely unknown. Notwithstanding the scarce and conflicting information available on this topic, the American Society of Clinical Oncology guidelines recommend sperm cryopreservation in males undergoing ICIs. As investigations regarding the long-term outcomes of anticancer immunotherapy on the male reproductive system are still in their infancy, this review aims to support and spur future research in order to understand a potential gonadotoxic effect of ICIs on testicular function, spermatogenesis and male fertility.

Ldha-Dependent Metabolic Programs in Sertoli Cells Regulate Spermiogenesis in Mouse Testis.

Sertoli cells play indispensable roles in spermatogenesis by providing the advanced germ cells with structural, nutritional, and regulatory support. Lactate is regarded as an essential Sertoli-cell-derived energy metabolite that nurses various types of spermatogenic cells; however, this assumption has not been tested using genetic approaches. Here, we have reported that the depletion of lactate production in Sertoli cells by conditionally deleting lactate dehydrogenase A (Ldha) greatly affected spermatogenesis. Ldha deletion in Sertoli cells significantly reduced the lactate production and resulted in severe defects in spermatogenesis. Spermatogonia and spermatocytes did not show even mild impairments, but the spermiogenesis of Ldha conditional knockout males was severely disrupted. Further analysis revealed that 2456 metabolites were altered in the sperm of the knockout animals, and specifically, lipid metabolism was dysregulated, including choline, oleic acid, and myristic acid. Surprisingly, choline supplementation completely rescued the spermiogenesis disorder that was caused by the loss of Ldha activities. Collectively, these data have demonstrated that the interruption of Sertoli-cell-derived lactate impacted sperm development through a choline-mediated mechanism. The outcomes of these findings have revealed a novel function of lactate in spermatogenesis and have therapeutic applications in treating human infertility.

Делеции в AZF локусе и нарушения сперматогенеза у мужчин c мозаицизмом по хромосоме Y

Введение. Аномалии кариотипа и микроделеции в локусе AZF (Yq11.2) хромосомы Y являются частыми генетическими причинами мужского бесплодия, вызванного нарушением сперматогенеза, однако эффект их сочетанного влияния недостаточно исследован. Цель: сравнительный анализ сперматологических показателей у мозаиков по хромосоме Y, имеющих и не имеющих делеции в локусе AZF. Методы. Обследованы 16 мужчин с мозаицизмом по хромосоме Y. Анализ кариотипа выполнен с использованием стандартного цитогенетического исследования на культивированных лимфоцитах периферической крови. Молекулярно-цитогенетическое (FISH) исследование проводили для выявления/верификации мозаицизма и структурных аномалий хромосомы Y. Микроделеции хромосомы Y детектировали методом мультиплексной ПЦР. Спермиологическое исследование выполняли в соответствии с рекомендациями Руководства ВОЗ (2010). Результаты. Цитогенетически идентифицируемые несбалансированные аномалии хромосомы Y обнаружены у 13 (81,25%) из 16 мужчин. Делеции в локусе AZF детектированы у 8 (50%) из 16 пациентов. Выборка разделена на две группы: группа I - без AZF делеций (n=8); и II - с AZF делециями (AZFb+c, n=7 и AZFc, n=1). Выявлены различия в структуре сперматологических диагнозов между группами: в группе I отмечены различные формы патозооспермии (азооспермия - 3; олигоастенотератозооспермия - 3; астенотератозооспермия - 2); в группе II - только тяжелые формы патозооспермии (азооспермия - 7; олигозооспермия тяжелой степени - 1), а также по частоте олигоспермии - 12,5% и 37,5%, соответственно. Более высокая концентрация сперматозоидов обнаружена у пациентов без AZF делеций (группа I - 15,9±31,0 млн/мл, группа II - 0,003±0,009 млн/мл; р=0,026). Не выявлено статистически значимых различий между группами по среднему возрасту пациентов, объёму, pH и вязкости эякулята. Выводы. Структурные аномалии и микроделеции хромосомы Y в локусе AZF (Yq11.2) встречаются с высокой частотой у пациентов с мозаицизмом по хромосоме Y. Несбалансированные перестройки хромосомы Y и патогенные микроделеции в локусе AZF у мужчин-мозаиков по хромосоме Y характеризуются тяжелой степенью нарушения сперматогенеза. Сохранение фертильностного потенциала мужчин с мозаицизмом по хромосоме Y возможно при отсутствии несбалансированных перестроек и тяжелых типов AZF делеций. Introduction. Chromosome abnormalities and microdeletions in the AZF (Yq11.2) locus of the Y chromosome are common genetic causes of male infertility caused by impaired spermatogenesis, however, the effect of their combined influence has not been sufficiently investigated. Aim: a comparative analysis of semen parameters in the Y-chromosome mosaics with and without AZF deletions. Methods. 16 male patients with Y chromosome mosaicism were examined. Chromosome analysis was done using by standard cytogenetic examination. Fluorescence in situ hybridization (FISH) was performed to identify/verify mosaicism and determine the structural Y chromosome abnormalities. The Y chromosome microdeletions were detected by multiplex PCR. The semen examination was carried out with the recommendations of the WHO Guidelines (2010). Results. Cytogenetically identifiable unbalanced the Y chromosome abnormalities were found in 13 patients (81.2%) of 16 patients. AZF deletion were detected in 8 patients (50%) of 16 patients. The patients were divided into two groups: group I - patients without AZF deletions (n=8), group II - patients with AZF deletions: AZFb+c, n=7 and AZFc (b2/b4), n=1 (n=8). Prominent difference in sperm diagnosis was revealed between the groups: in group I, there was a various degree of pathozoospermia (azoospermia - 3; oligoastenoteratozoospermia - 3; asthenoteratozoospermia - 2); in group II, only severe forms of pathozoospermia (azoospermia - 7; oligozoospermia severe - 1), as well as the frequency of oligospermia - 12.5% and 37.5%, respectively. A higher concentration of spermatozoa was found in patients without AZF deletions (group I - 15.9±31.0 million/ml, group II - 0.003±0.009 million/ml; p=0.026). There was no statistically significant difference between the average age of patients in the groups, ejaculate volume, pH and viscosity of ejaculate. Conclusion. There is high frequency of structural Y chromosome abnormalities and microdeletions in the AZF (Yq11.2) locus in the Y chromosome mosaics. Unbalanced cytogenetic Y chromosome rearrangements and pathogenic AZF microdeletions are characterized by a severe degree of spermatogenesis disorder in male patients with Y chromosome mosaicism. The preservation of fertility potential in men with Y chromosome mosaicism is possible in the absence of unbalanced rearrangements and severe types of AZF deletions.

Absence of Testicular Estrogen Leads to Defects in Spermatogenesis and Increased Semen Abnormalities in Male Rabbits.

Estrogens are steroid hormones produced by the aromatization of androgens by the aromatase enzyme, encoded by the CYP19A1 gene. Although generally referred to as "female sex hormones", estrogen is also produced in the adult testes of many mammals, including humans. To better understand the function of estrogens in the male, we used the rabbit model which is an important biomedical model. First, the expression of CYP19A1 transcripts was localized mainly in meiotic germ cells. Thus, testicular estrogen appears to be produced inside the seminiferous tubules. Next, the cells expressing ESR1 and ESR2 were identified, showing that estrogens could exert their function on post-meiotic germ cells in the tubules and play a role during sperm maturation, since ESR1 and ESR2 were detected in the cauda epididymis. Then, CRISPR/Cas9 CYP19A1-/- genetically modified rabbits were analyzed. CYP19A1-/- males showed decreased fertility with lower sperm count associated with hypo-spermatogenesis and lower spermatid number. Germ/sperm cell DNA methylation was unchanged, while sperm parameters were affected as CYP19A1-/- males exhibited reduced sperm motility associated with increased flagellar defects. In conclusion, testicular estrogens could be involved in the spermatocyte-spermatid transition in the testis, and in the acquisition of sperm motility in the epididymis.

PMON33 Non-Syndromic SRY Negative 46 XX Testicular Disorder of Sex Development (DSD): A Rare Cause of Infertility

Abstract Introduction Male infertility can be categorized as endocrine and systemic disorders, primary testicular defects in spermatogenesis, sperm transport disorders and idiopathic male infertility. Chromosomal abnormalities are one of the primary testicular defects. The overall incidence of chromosome abnormalities in infertile men is estimated to be around 5.8%. XX male is a rare sex chromosomal disorder in infertile men. We present case of a young patient with infertility, azoospermia, hypergonadotropic hypogonadism and classical (46, XX) karyotype with negative FISH and SRY gene. Case report: 28-Year-old gentleman was referred to adult endocrinology clinic as part of work up for infertility. Initial work up included 2 semen analyses which were consistent with azoospermia. Examination revealed height 180 cm, weight 77 kg, a body mass index 24 kg/m2, normal phenotypic male without gynecomastia and had normal male pattern hair distribution. Genital examination showed normal male phallus without hypospadias, a hypoplastic scrotum, bilateral firm testes, with no varicocele or hydrocele. Labs were suggestive of compensated primary hypogonadism (FSH 47.9 mIU/ml, LH 20.4 mIU/ml, total testosterone 397.9 ng/dl by LC/MS, free testosterone 15.76 ng/dl by equilibrium dialysis, Inhibin B < 10 pg/ml, and anti-Mullerian hormone 0.390 ng/ml. Given normal testosterone levels, he was not started on testosterone replacement. Karyotype analysis revealed 46 XX and FISH was negative for SRY gene. CT imaging of abdomen and pelvis did not reveal any Mullerian structures, prostrate and seminal vesicles reported as normal. Patient met with a Urologist and was informed that testicular sperm extraction will not be a possibility for him. Patient had a detailed consultation with a geneticist for genetic and family counseling. Chromosome microarray analysis was obtained and was normal. Invitae Disorders of sex development (DSD) panel obtained for genetic sequencing of 53 genes including SOX-9, DHH, WNT4, WT1, was found to be non-diagnostic. Conclusion and Discussion SRY negative 46 XX male is a DSD and a rare genetic cause of male factor infertility, with a discrepancy between genotype and phenotypic sex. In the case presented, genetic work up for potential etiologies has been unrevealing. Further studies are needed to identify the genetic causes of the SRY negative 46 XX male phenotype. Physicians involved in care of these patients should orient with clinical management and prognosis. For Classical 46 XX Males seeking fertility, the current options include invitro-fertilization with donor sperm or adoption only. Testosterone replacement should be considered to correct hypogonadism for physical and sexual well-being. Genetic consultation should be sought in all these cases. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

PMON33 Non-Syndromic SRY Negative 46 XX Testicular Disorder of Sex Development (DSD); A rare cause of Infertility

Abstract Introduction Male infertility can be categorized as endocrine and systemic disorders, primary testicular defects in spermatogenesis, sperm transport disorders and idiopathic male infertility. Chromosomal abnormalities are one of the primary testicular defects. The overall incidence of chromosome abnormalities in infertile men is estimated to be around 5.8%. XX male is a rare sex chromosomal disorder in infertile men. We present case of a young patient with infertility, azoospermia, hypergonadotropic hypogonadism and classical (46, XX) karyotype with negative FISH and SRY gene. Case report: 28-Year-old gentleman was referred to adult endocrinology clinic as part of work up for infertility. Initial work up included 2 semen analyses which were consistent with azoospermia. Examination revealed height 180 cm, weight 77 kg, a body mass index 24 kg/m2, normal phenotypic male without gynecomastia and had normal male pattern hair distribution. Genital examination showed normal male phallus without hypospadias, a hypoplastic scrotum, bilateral firm testes, with no varicocele or hydrocele. Labs were suggestive of compensated primary hypogonadism (FSH 47.9 mIU/ml, LH 20.4 mIU/ml, total testosterone 397.9 ng/dl by LC/MS, free testosterone 15.76 ng/dl by equilibrium dialysis, Inhibin B < 10 pg/ml, and anti-Mullerian hormone 0.390 ng/ml. Given normal testosterone levels, he was not started on testosterone replacement. Karyotype analysis revealed 46 XX and FISH was negative for SRY gene. CT imaging of abdomen and pelvis did not reveal any Mullerian structures, prostrate and seminal vesicles reported as normal. Patient met with a Urologist and was informed that testicular sperm extraction will not be a possibility for him. Patient had a detailed consultation with a geneticist for genetic and family counseling. Chromosome microarray analysis was obtained and was normal. Invitae Disorders of sex development (DSD) panel obtained for genetic sequencing of 53 genes including SOX-9, DHH, WNT4, WT1, was found to be non-diagnostic. Conclusion and Discussion SRY negative 46 XX male is a DSD and a rare genetic cause of male factor infertility, with a discrepancy between genotype and phenotypic sex. In the case presented, genetic work up for potential etiologies has been unrevealing. Further studies are needed to identify the genetic causes of the SRY negative 46 XX male phenotype. Physicians involved in care of these patients should orient with clinical management and prognosis. For Classical 46 XX Males seeking fertility, the current options include invitro-fertilization with donor sperm or adoption only. Testosterone replacement should be considered to correct hypogonadism for physical and sexual well-being. Genetic consultation should be sought in all these cases. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The function of Foxo1 in spermatogonia development is independent of PI3K/PTEN signaling

Spermatogenesis is a highly coordinated process that initiates shortly after birth and continues throughout the lifespan of male animals. Foxo1 is a transcription factor and is involved in many biological processes. It has been reported that the inactivation of Foxo1 in gonocytes during the embryonic stage causes the defects of spermatogenesis. In the present study, we found that the inactivation of Foxo1 in spermatogonia after birth also caused germ cell loss and male infertility. We found that the initiation of meiosis was not affected; however, the germ cell development was arrested after meiosis and lack of mature spermatozoa in the cauda epididymis. We also found that the proliferation of Foxo1-deficient spermatogonia stem cells was significantly reduced under in vitro conditions. Further study revealed that inactivation of Pten in postnatal spermatogonia using Stra8-Cre did not affect germ cell development and the subcellular location of FOXO1 in Pten-deficient spermatogonia. This study demonstrated that Foxo1 was involved in the development of spermatogonia after birth and the function of Foxo1 was probably not regulated by PI3K/PTEN signaling.

Mutation in Drosophila concentrative nucleoside transporter 1 alters spermatid maturation and mating behavior.

Concentrative nucleoside transporters (Cnts) are unidirectional carriers that mediate the energy-costly influx of nucleosides driven by the transmembrane sodium gradient. Cnts are transmembrane proteins that share a common structural organization and are found in all phyla. Although there have been studies on Cnts from a biochemical perspective, no deep research has examined their role at the organismal level. Here, we investigated the role of the Drosophila melanogaster cnt1 gene, which is specifically expressed in the testes. We used the CRISPR/Cas9 system to generate a mutation in the cnt1 gene. The cnt1 mutants exhibited defects in the duration of copulation and spermatid maturation, which significantly impaired male fertility. The most striking effect of the cnt1 mutation in spermatid maturation was an abnormal structure of the sperm tail, in which the formation of major and minor mitochondrial derivatives was disrupted. Our results demonstrate the importance of cnt1 in male fertility and suggest that the observed defects in mating behavior and spermatogenesis are due to alterations in nucleoside transport and associated metabolic pathways.

Glutathione S-transferase Mu 3 is associated to in vivo fertility, but not sperm quality, in bovine

In the dairy breeding industry, pregnancy of dairy cows is essential to initiate milk production, so that high fertility rates are required to increase their productivity. In this regard, sperm proteins that are indicative of sperm quality and/or fertility have become an important target of study. Glutathione S-transferase Mu 3 (GSTM3) has been established as a fertility and sperm quality parameter in humans and pigs and, consequently, it might be a potential biomarker in cattle. For this reason, the present work aimed to determine if GSTM3 could predict sperm quality and in vivo fertility in this species. Sperm quality was assessed with flow cytometry and computer-assisted sperm analysis. Immunoblotting and immunofluorescence analysis were performed to determine the presence and localisation pattern of sperm GSTM3. This enzyme was found to be present in bovine sperm and to be localised along the sperm tail and the equatorial segment of the head. No significant associations between sperm GSTM3 and sperm quality parameters were observed, except a negative association with morphologically abnormal sperm having a coiled tail. In addition, and more relevant, higher levels of GSTM3 in sperm were seen in bulls showing lower in vivo fertility rates. In conclusion, our data evidenced the presence of GSTM3 in bovine sperm. Moreover, we suggest that, despite not being associated with sperm quality, GSTM3 might be an in vivo subfertility biomarker in cattle sperm, and that high levels of this protein could be an indicative of defective spermatogenesis and/or epididymal maturation.

Ankle2 deficiency-associated microcephaly and spermatogenesis defects in zebrafish are alleviated by heterozygous deletion of vrk1

Autosomal recessive primary microcephaly (MCPH) is a rare congenital disorder characterized by a below average brain volume at birth and is associated with neurodevelopmental disorders such as growth retardation and intellectual disability. Mutations in ANKLE2 have been identified as one of the causes of MCPH (MCPH16). ANKLE2 is a target molecule of the Zika virus NS4a protein that interferes with ANKLE2 function, resulting in severe microcephaly. ANKLE2 is essential for organizing the nuclear envelope and chromatin structures during the mitotic-end process via barrier to autointegration factor (BAF) dephosphorylation. However, the precise mechanism by which the loss of ANKLE2 function causes the pathogenesis of microcephaly remains unclear. In this study, we generated Ankle2-deficient zebrafish (ankle2−/−) with a significant reduction in brain size compared with that of their control siblings. The ankle2−/− brain showed a significant decrease in the number of radial glial progenitor cells, suggesting that Ankle2 deficiency in zebrafish causes neurogenesis defects. Furthermore, ankle2−/− male zebrafish showed infertility owing to defects in spermatogenesis. Notably, microcephaly was overcome by vrk1 morpholino knockdown or vrk1 heterozygous deletion. In addition, spermatogenesis in ankle2−/− zebrafish males was partially restored by the vrk1 heterozygous deletion, although infertility was not resolved. These results indicate that ANKLE2 and VRK1 coordinate with each other for BAF phosphorylation to maintain normal mitosis during neurogenesis and spermatogenesis.

Genetic analysis of Caenorhabditis elegans Haspin-like genes shows that hasp-1 plays multiple roles in the germline.

ABSTRACTHaspin is a histone kinase that promotes error-free chromosome segregation by recruiting the chromosomal passenger complex (CPC) to mitotic and meiotic chromosomes. Haspin remains less well studied than other M-phase kinases, and the models explaining Haspin function have been developed primarily in mitotic cells. Here, we generate strains containing new conditional or nonsense mutations in the Caenorhabditis elegans Haspin homologs hasp-1 and hasp-2 and characterize their phenotypes. We show that hasp-1 is responsible for all predicted functions of Haspin and that loss of function of hasp-1 using classical and conditional alleles produces defects in germline stem cell proliferation and spermatogenesis, and confirms its role in oocyte meiosis. Genetic analysis suggests that hasp-1 acts downstream of the Polo-like kinase plk-2 and shows synthetic interactions between hasp-1 and two genes expected to promote recruitment of the CPC by a parallel pathway that depends on the kinase Bub1. This work adds to the growing understanding of Haspin function by characterizing a variety of roles in an intact animal.

Effects of PRP Intratesticle Injection in Non-Obstructive Azoospermic Infertile Men in vivo on Spermatogenesis and Reproductive Hormonal Levels(FSH and LH)

Non-obstructive azoospermia (NOA) is the absence of sperm in infertile men ejaculate as a result of significant spermatogenesis defects or hormonal malfunction. PRP intratesticular injection may be employed in assisted reproductive technology as a therapeutic effect for this problem. The aim is to assess the level of spermatogenesis and reproductive hormones after PRP autologous intratesticular injection. 50 nonabstructive azoospermia infertile men participated in the current study. The patients were given an intra-testicular injection of platelet-rich plasma using a 1 ml syringe; FSH LH hormonal levels were estimated; if there was no sperm count, the patient should be prepared for a testicular biopsy by the expert doctor. Small number of sperm may be discovered during a biopsy while the testes are being examined in the lab. The therapeutic benefits were seen in 15 patients 3-4 months after using autologous platelet-rich plasma. The results showed that there was a highly significant difference in FSH and LH hormonal levels after PRP injection P(0.001). PRP can help non-obstructive azoospermic infertile males with their testis' structural and functional dysfunction.

Disruption of cell division prevents gametogenesis in triploid Pacific oysters (Crassostrea gigas)

Triploid oysters with their large size and consistent meat quality have become a major component of the oyster aquaculture industry worldwide. It has been observed that triploid Pacific oysters (Crassostrea gigas) showed high levels of variation and sex-specific differences in sterility. Here, we analyzed mitosis and meiosis during gametogenesis in triploid Pacific oysters and the expression of key cell cycle regulatory proteins, namely members of the cyclin family (A, B, D, E). We found that in female C. gigas, expression of cyclin D and E in gonad of triploids were higher than those in diploids, while expression of cyclin A and B were significantly suppressed in the gonads of triploids. To test whether mitosis of gonia might be arrested at G1/S or S/G2 phase in triploids, we compared EdU labeling in gonadal cells of triploid and diploid oysters. The results showed a similar number of EdU-labelled cells in gonads of triploid and diploid females, indicating that gonia in triploids successfully entered the S-phase as diploids. In male triploids, gonadal expression of the four cyclin genes at the mature stage were similar to that in male diploids at the growth stage with numerous spermatogenic cells. The number of EdU-labelled cells in triploid male gonads at the mature stage was significantly more than that in diploids, suggesting more proliferating cells in triploid gonads in males. Collectively, these data inidate that the sterility of triploid females is likely caused by the failure of germ cells to enter G2-phase during mitosis. In addition, we speculate that gonadal development of triploid males was arrested at the growth stage and defective spermatogenesis might be associated with disruption of meiosis.

Sperm-specific glycogen synthase kinase 3 is required for sperm motility and the post-fertilization signal for female meiosis II in Caenorhabditis elegans.

In most sexually reproducing animals, sperm entry provides the signal to initiate the final stages of female meiosis. In Caenorhabditis elegans, this signal is required for completion of female anaphase I and entry into meiosis II (MII). memi-1/2/3 (meiosis-to-mitosis) encode maternal components that facilitate this process; memi-1/2/3(RNAi) results in a skipped-MII phenotype. Previously, we used a gain-of-function mutation, memi-1(sb41), to identify genetic suppressors that represent candidates for the sperm-delivered signal. Herein, we characterize two suppressors of memi-1(sb41): gskl-1 and gskl-2. Both genes encode functionally redundant sperm glycogen synthase kinase, type 3 (GSK3) protein kinases. Loss of both genes causes defects in male spermatogenesis, sperm pseudopod treadmilling and paternal-effect embryonic lethality. The two kinases locate within the pseudopod of activated sperm, suggesting that they directly or indirectly regulate the sperm cytoskeletal polymer major sperm protein (MSP). The GSK3 genes genetically interact with another memi-1(sb41) suppressor, gsp-4, which encodes a sperm-specific PP1 phosphatase, previously proposed to regulate MSP dynamics. Moreover, gskl-2 gsp-4; gskl-1 triple mutants often skip female MII, similar to memi-1/2/3(RNAi). The GSK3 kinases and PP1 phosphatases perform similar sperm-related functions and work together for post-fertilization functions in the oocyte that involve MEMI.

PD36-05 LOSS-OF-FUNCTION IN PHF7 CAUSE MALE INFERTILITY BY IMPAIRING HISTONE-TO-PROTAMINE EXCHANGE DURING SPERMIOGENESIS

You have accessJournal of UrologyCME1 May 2022PD36-05 LOSS-OF-FUNCTION IN PHF7 CAUSE MALE INFERTILITY BY IMPAIRING HISTONE-TO-PROTAMINE EXCHANGE DURING SPERMIOGENESIS Jianxing Cheng, Mengyang Cao, Zhongjie Zheng, and Haocheng Lin Jianxing ChengJianxing Cheng More articles by this author , Mengyang CaoMengyang Cao More articles by this author , Zhongjie ZhengZhongjie Zheng More articles by this author , and Haocheng LinHaocheng Lin More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002594.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Introduction: Recently, studies shows that PHF7 is specifically expressed in elongating spermatid during spermiogenesis, however whether it results infertility in human are still unclear as well as the mechanism. Objective: To investigate the role of PHF7 on male infertility and explore its mechanism. METHODS: The blood and testicular samples from azoospermia patients were collected and used for genome sequencing and immunohistology. The PHF7 knockout mice model were established and used for animal study. RT-PCR, Immunohistology, GST-pulldown and western blotting were used for mechanism exploring. RESULTS: Our results revealed that expression level of PHF7 in patients with male infertility was significant less than control. Two heterozygous mutations were also identified in azoospemia patients. PHF7 knockout mice show defective spermatogenesis at a later spermiogenic stage with abnormal sperm morphology with histone retention. CONCLUSIONS: Our study identified PHF7 mutations as etiological factors in male infertility and reveal its mechanism for regulating the histone-to-protamine exchange during spermiogenesis. Source of Funding: Peking University Clinical Medicine +X Youth Special Program (PKU2018LCXQ019) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e634 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jianxing Cheng More articles by this author Mengyang Cao More articles by this author Zhongjie Zheng More articles by this author Haocheng Lin More articles by this author Expand All Advertisement PDF DownloadLoading ...

Predicting male fertility from the sperm methylome: application to 120 bulls with hundreds of artificial insemination records

BackgroundConflicting results regarding alterations to sperm DNA methylation in cases of spermatogenesis defects, male infertility and poor developmental outcomes have been reported in humans. Bulls used for artificial insemination represent a relevant model in this field, as the broad dissemination of bull semen considerably alleviates confounding factors and enables the precise assessment of male fertility. This study was therefore designed to assess the potential for sperm DNA methylation to predict bull fertility.ResultsA unique collection of 100 sperm samples was constituted by pooling 2–5 ejaculates per bull from 100 Montbéliarde bulls of comparable ages, assessed as fertile (n = 57) or subfertile (n = 43) based on non-return rates 56 days after insemination. The DNA methylation profiles of these samples were obtained using reduced representation bisulfite sequencing. After excluding putative sequence polymorphisms, 490 fertility-related differentially methylated cytosines (DMCs) were identified, most of which were hypermethylated in subfertile bulls. Interestingly, 46 genes targeted by DMCs are involved in embryonic and fetal development, sperm function and maturation, or have been related to fertility in genome-wide association studies; five of these were further analyzed by pyrosequencing. In order to evaluate the prognostic value of fertility-related DMCs, the sperm samples were split between training (n = 67) and testing (n = 33) sets. Using a Random Forest approach, a predictive model was built from the methylation values obtained on the training set. The predictive accuracy of this model was 72% on the testing set and 72% on individual ejaculates collected from an independent cohort of 20 bulls.ConclusionThis study, conducted on the largest set of bull sperm samples so far examined in epigenetic analyses, demonstrated that the sperm methylome is a valuable source of male fertility biomarkers. The next challenge is to combine these results with other data on the same sperm samples in order to improve the quality of the model and better understand the interplay between DNA methylation and other molecular features in the regulation of fertility. This research may have potential applications in human medicine, where infertility affects the interaction between a male and a female, thus making it difficult to isolate the male factor.