Actin-related protein ACTL7B ablation leads to OAT with multiple morphological abnormalities of the flagellum and male infertility in mice†.

The formation of fertilisation-competent sperm requires spermatid morphogenesis (spermiogenesis), a poorly understood program that involves complex coordinated restructuring and specialised cytoskeletal structures. A major class of cytoskeletal regulators are the actin-related proteins (ARPs), which include conventional actin variants, and related proteins that play essential roles in complexes regulating actin dynamics, intracellular transport, and chromatin remodeling. Multiple testis-specific ARPs are well conserved among mammals, but their functional roles are unknown. One of these is actin-like 7b (Actl7b) that encodes an orphan ARP highly similar to the ubiquitously expressed beta actin (ACTB). Here we report ACTL7B is expressed in human and mouse spermatids through the elongation phase of spermatid development. In mice, ACTL7B specifically localises to the developing acrosome, within the nucleus of early spermatids, and to the flagellum connecting region. Based on this localisation pattern and high level of sequence conservation in mice, humans, and other mammals, we examined the requirement for ACTL7B in spermiogenesis by generating and characterising the reproductive phenotype of male Actl7b KO mice. KO mice were infertile, with severe and variable oligoteratozoospermia (OAT) and multiple morphological abnormalities of the flagellum (MMAF) and sperm head. These defects phenocopy human OAT and MMAF, which are leading causes of idiopathic male infertility. In conclusion, this work identifies ACTL7B as a key regulator of spermiogenesis that is required for male fertility.

Germ Cell-Specific Retinoic Acid Receptor α Functions in Germ Cell Organization, Meiotic Integrity, and Spermatogonia.

Retinoic acid receptor α (RARA), a retinoic acid-dependent transcription factor, is expressed in both somatic and germ cells of the testis. Rara-null male mice with global Rara mutations displayed severely degenerated testis and infertility phenotypes. To elucidate the specific responsibility of germ cell RARA in spermatogenesis, Rara was deleted in germ cells, generating germ cell-specific Rara conditional knockout (cKO) mice. These Rara cKO animals exhibited phenotypes of quantitatively reduced epididymal sperm counts and disorganized germ cell layers in the seminiferous tubules, which worsened with aging. Abnormal tubules lacked lumen, contained vacuoles, and showed massive germ cell sloughing, all characteristics similar to those observed in Rara-null tubules. Spermatocyte chromosomal spreads revealed a novel role for germ cell RARA in modulating the integrity of synaptonemal complexes and meiotic progression. Furthermore, the initiation of spermatogenesis from spermatogonial stem cells was decreased in Rara cKO testes following busulfan treatment, supporting a role of germ cell RARA in spermatogonial proliferation. Collectively, the evidence in this study indicates that RARA produced in male germ cells has a broad spectrum of functions throughout spermatogenesis, which includes the maintenance of seminiferous epithelium organization, the integrity of the meiotic genome, and spermatogonial proliferation and differentiation. The results further suggest that germ cell RARA has dual functions: intrinsically in germ cells, balancing proliferation and differentiation of spermatogonia, and controlling genome integrity during meiosis; and extrinsically in the crosstalks with Sertoli cells, controlling the cell junctional physiology for coordinating proper spatial and temporal development of germ cells during spermatogenesis.

SPATC1L maintains the integrity of the sperm head-tail junction.

Spermatogenesis is a tightly regulated process involving germ cell-specific and germ cell-predominant genes. Here we investigate a novel germ cell-specific gene, Spatc1l (spermatogenesis and centriole associated 1 like). Expression analyses show that SPATC1L is expressed in mouse and human testes. We find that mouse SPATC1L localizes to the neck region in testicular sperm. Moreover, SPATC1L associates with the regulatory subunit of protein kinase A (PKA). Using CRISPR/Cas9-mediated genome engineering, we generate mice lacking SPATC1L. Disruption of Spatc1l in mice leads to male sterility owing to separation of sperm heads from tails. The lack of SPATC1L is associated with a reduction in PKA activity in testicular sperm, and we identify capping protein muscle Z-line beta as a candidate target of phosphorylation by PKA in testis. Taken together, our results implicate the SPATC1L-PKA complex in maintaining the stability of the sperm head-tail junction, thereby revealing a new molecular basis for sperm head-tail integrity.

Rhox13 is required for a quantitatively normal first wave of spermatogenesis in mice.

We previously described a novel germ cell-specific X-linked reproductive homeobox gene (Rhox13) that is upregulated at the level of translation in response to retinoic acid (RA) in differentiating spermatogonia and preleptotene spermatocytes. We hypothesize that RHOX13 plays an essential role in male germ cell differentiation, and have tested this by creating a Rhox13 gene knockout (KO) mouse. Rhox13 KO mice are born in expected Mendelian ratios, and adults have slightly reduced testis weights, yet a full complement of spermatogenic cell types. Young KO mice (at ~7-8 weeks of age) have a ≈50% reduction in epididymal sperm counts, but numbers increased to WT levels as the mice reach ~17 weeks of age. Histological analysis of testes from juvenile KO mice reveals a number of defects during the first wave of spermatogenesis. These include increased apoptosis, delayed appearance of round spermatids and disruption of the precise stage-specific association of germ cells within the seminiferous tubules. Breeding studies reveal that both young and aged KO males produce normal-sized litters. Taken together, our results indicate that RHOX13 is not essential for mouse fertility in a controlled laboratory setting, but that it is required for optimal development of differentiating germ cells and progression of the first wave of spermatogenesis.

Transcription Factor GLIS3: A New and Critical Regulator of Postnatal Stages of Mouse Spermatogenesis.

In this study, we identify a novel and essential role for the Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in the regulation of postnatal spermatogenesis. We show that GLIS3 is expressed in gonocytes, spermatogonial stem cells (SSCs) and spermatogonial progenitors (SPCs), but not in differentiated spermatogonia and later stages of spermatogenesis or in somatic cells. Spermatogenesis is greatly impaired in GLIS3 knockout mice. Loss of GLIS3 function causes a moderate reduction in the number of gonocytes, but greatly affects the generation of SSCs/SPCs, and as a consequence the development of spermatocytes. Gene expression profiling demonstrated that the expression of genes associated with undifferentiated spermatogonia was dramatically decreased in GLIS3-deficient mice and that the cytoplasmic-to-nuclear translocation of FOXO1, which marks the gonocyte-to-SSC transition and is necessary for SSC self-renewal, is inhibited. These observations suggest that GLIS3 promotes the gonocyte-to-SSC transition and is a critical regulator of the dynamics of early postnatal spermatogenesis. Stem Cells 2016;34:2772-2783.

Male infertility in mice lacking the store-operated Ca(2+) channel Orai1.

Store-operated calcium entry (SOCE) is an important Ca(2+) influx pathway in somatic cells. In addition to maintaining endoplasmic reticulum (ER) Ca(2+) stores, Ca(2+) entry through store-operated channels regulates essential signaling pathways in numerous cell types. Patients with mutations in the store-operated channel subunit ORAI1 exhibit defects in store-operated Ca(2+) influx, along with severe immunodeficiency, congenital myopathy and ectodermal dysplasia. However, little is known about the functional role of ORAI1 in germ cells and reproductive function in mice, or in men, since men with loss-of-function or null mutations in ORAI1 rarely survive to reproductive age. In this study, we investigated the role of ORAI1 in male reproductive function. We reveal that Orai1(-/-) male mice are sterile and have severe defects in spermatogenesis, with prominent deficiencies in mid- to late-stage elongating spermatid development. These studies establish an essential in vivo role for store-operated ORAI1 channels in male reproductive function and identify these channels as potential non-steroidal regulators of male fertility.

Targeting the Gdnf Gene in peritubular myoid cells disrupts undifferentiated spermatogonial cell development.

Spermatogonial stem cells (SSCs) are a subpopulation of undifferentiated spermatogonia located in a niche at the base of the seminiferous epithelium delimited by Sertoli cells and peritubular myoid (PM) cells. SSCs self-renew or differentiate into spermatogonia that proliferate to give rise to spermatocytes and maintain spermatogenesis. Glial cell line-derived neurotrophic factor (GDNF) is essential for this process. Sertoli cells produce GDNF and other growth factors and are commonly thought to be responsible for regulating SSC development, but limited attention has been paid to the role of PM cells in this process. A conditional knockout (cKO) of the androgen receptor gene in PM cells resulted in male infertility. We found that testosterone (T) induces GDNF expression in mouse PM cells in vitro and neonatal spermatogonia (including SSCs) co-cultured with T-treated PM cells were able to colonize testes of germ cell-depleted mice after transplantation. This strongly suggested that T-regulated production of GDNF by PM cells is required for spermatogonial development, but PM cells might produce other factors in vitro that are responsible. In this study, we tested the hypothesis that production of GDNF by PM cells is essential for spermatogonial development by generating mice with a cKO of the Gdnf gene in PM cells. The cKO males sired up to two litters but became infertile due to collapse of spermatogenesis and loss of undifferentiated spermatogonia. These studies show for the first time, to our knowledge, that the production of GDNF by PM cells is essential for undifferentiated spermatogonial cell development in vivo.

Disrupting Cyclin Dependent Kinase 1 in Spermatocytes Causes Late Meiotic Arrest and Infertility in Mice.

While cyclin dependent kinase 1 (CDK1) has a critical role in controlling resumption of meiosis in oocytes, its role has not been investigated directly in spermatocytes. Unique aspects of male meiosis led us to hypothesize that its role is different in male meiosis than in female meiosis. We generated a conditional knockout (cKO) of the Cdk1 gene in mouse spermatocytes to test this hypothesis. We found that CDK1-null spermatocytes undergo synapsis, chiasmata formation, and desynapsis as is seen in oocytes. Additionally, CDK1-null spermatocytes relocalize SYCP3 to centromeric foci, express H3pSer10, and initiate chromosome condensation. However, CDK1-null spermatocytes fail to form condensed bivalent chromosomes in prophase of meiosis I and instead are arrested at prometaphase. Thus, CDK1 has an essential role in male meiosis that is consistent with what is known about the role of CDK1 in female meiosis, where it is required for formation of condensed bivalent metaphase chromosomes and progression to the first meiotic division. We found that cKO spermatocytes formed fully condensed bivalent chromosomes in the presence of okadaic acid, suggesting that cKO chromosomes are competent to condense, although they do not do so in vivo. Additionally, arrested cKO spermatocytes exhibited irregular cell shape, irregular large nuclei, and large distinctive nucleoli. These cells persist in the seminiferous epithelium through the next seminiferous epithelial cycle with a lack of stage XII checkpoint-associated cell death. This indicates that CDK1 is required upstream of a checkpoint-associated cell death as well as meiotic metaphase progression in mouse spermatocytes.

Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes.

Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co-localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post-meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2-GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post-meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage-specific spermatogenic cell proteins and developmental events.

Peritubular myoid cells participate in male mouse spermatogonial stem cell maintenance.

Peritubular myoid (PM) cells surround the seminiferous tubule and together with Sertoli cells form the cellular boundary of the spermatogonial stem cell (SSC) niche. However, it remains unclear what role PM cells have in determining the microenvironment in the niche required for maintenance of the ability of SSCs to undergo self-renewal and differentiation into spermatogonia. Mice with a targeted disruption of the androgen receptor gene (Ar) in PM cells experienced a progressive loss of spermatogonia, suggesting that PM cells require testosterone (T) action to produce factors influencing SSC maintenance in the niche. Other studies showed that glial cell line-derived neurotrophic factor (GDNF) is required for SSC self-renewal and differentiation of SSCs in vitro and in vivo. This led us to hypothesize that T-regulated GDNF expression by PM cells contributes to the maintenance of SSCs. This hypothesis was tested using an adult mouse PM cell primary culture system and germ cell transplantation. We found that T induced GDNF expression at the mRNA and protein levels in PM cells. Furthermore, when thymus cell antigen 1-positive spermatogonia isolated from neonatal mice were cocultured with PM cells with or without T and transplanted to the testes of germ cell-depleted mice, the number and length of transplant-derived colonies was increased considerably by in vitro T treatment. These results support the novel hypothesis that T-dependent regulation of GDNF expression in PM cells has a significant influence on the microenvironment of the niche and SSC maintenance.

Nuclear removal during terminal lens fiber cell differentiation requires CDK1 activity: appropriating mitosis-related nuclear disassembly.

Lens epithelial cells and early lens fiber cells contain the typical complement of intracellular organelles. However, as lens fiber cells mature they must destroy their organelles, including nuclei, in a process that has remained enigmatic for over a century, but which is crucial for the formation of the organelle-free zone in the center of the lens that assures clarity and function to transmit light. Nuclear degradation in lens fiber cells requires the nuclease DNase IIß (DLAD) but the mechanism by which DLAD gains access to nuclear DNA remains unknown. In eukaryotic cells, cyclin-dependent kinase 1 (CDK1), in combination with either activator cyclins A or B, stimulates mitotic entry, in part, by phosphorylating the nuclear lamin proteins leading to the disassembly of the nuclear lamina and subsequent nuclear envelope breakdown. Although most post-mitotic cells lack CDK1 and cyclins, lens fiber cells maintain these proteins. Here, we show that loss of CDK1 from the lens inhibited the phosphorylation of nuclear lamins A and C, prevented the entry of DLAD into the nucleus, and resulted in abnormal retention of nuclei. In the presence of CDK1, a single focus of the phosphonuclear mitotic apparatus is observed, but it is not focused in CDK1-deficient lenses. CDK1 deficiency inhibited mitosis, but did not prevent DNA replication, resulting in an overall reduction of lens epithelial cells, with the remaining cells possessing an abnormally large nucleus. These observations suggest that CDK1-dependent phosphorylations required for the initiation of nuclear membrane disassembly during mitosis are adapted for removal of nuclei during fiber cell differentiation.

Analysis of signaling pathways controlling flagellar movements in mammalian spermatozoa.

The mammalian sperm flagellum is an example of organelles where sensory and signaling functions are integrated with motility. Structurally related to other ciliary appendages, it has unique cytoskeletal structures that serve to assemble signaling complexes as well as components of metabolic pathways. Flagellar motility is regulated by signaling events that control sperm ion milieu, energy production, and classical second messenger-dependent phosphorylation cascades. Here, we will concentrate on the cAMP signaling pathway associated with flagellar motility. We will describe methods to analyze the properties of a unique adenylyl cyclase termed sAC (gene name Sacy, Adcy10), which plays an essential function in cAMP accumulation in spermatozoa. This soluble adenylyl cyclase (sAC) is a sensor for bicarbonate, pH, and Ca(2+) and is likely involved in coupling energy homeostasis with signaling events. We will also describe methods to investigate the macromolecular complexes that bring together sAC and other signaling molecules.

Glycolysis and mitochondrial respiration in mouse LDHC-null sperm.

We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable reduction in sperm glucose consumption, ATP production, and motility. While that study used mice with a mixed genetic background, the present study used C57BL/6 (B6) and 129S6 (129) Ldhc KO mice. We found that B6 KO males were subfertile and 129 KO males were infertile. Sperm from 129 wild-type (WT) mice have a lower glycolytic rate than sperm from B6 WT mice, resulting in a greater reduction in ATP production in 129 KO sperm than in B6 KO sperm. The lower glycolytic rate in 129 sperm offered a novel opportunity to examine the role of mitochondrial respiration in sperm ATP production and motility. We observed that in media containing a mitochondrial substrate (pyruvate or lactate) as the sole energy source, ATP levels and progressive motility in 129 KO sperm were similar to those in 129 WT sperm. However, when glucose was added, lactate was unable to maintain ATP levels or progressive motility in 129 KO sperm. The rate of respiration (ZO2) was high when 129 KO or WT sperm were incubated with lactate alone, but addition of glucose caused a reduction in ZO2. These results indicate that in the absence of glucose, 129 sperm can produce ATP via oxidative phosphorylation, but in the presence of glucose, oxidative phosphorylation is suppressed and the sperm utilize aerobic glycolysis, a phenomenon known as the Crabtree effect.

Disruption of a spermatogenic cell-specific mouse enolase 4 (eno4) gene causes sperm structural defects and male infertility.

Sperm utilize glycolysis to generate ATP required for motility, and several spermatogenic cell-specific glycolytic isozymes are associated with the fibrous sheath (FS) in the principal piece of the sperm flagellum. We used proteomics and molecular biology approaches to confirm earlier reports that a novel enolase is present in mouse sperm. We then found that a pan-enolase antibody, but not antibodies to ENO2 and ENO3, recognized a protein in the principal piece of the mouse sperm flagellum. Database analyses identified two previously uncharacterized enolase family-like candidate genes, 64306537H0Rik and Gm5506. Northern analysis indicated that 64306537H0Rik (renamed Eno4) was transcribed in testes of mice by Postnatal Day 12. To determine the role of ENO4, we generated mice using embryonic stem cells in which an Eno4 allele was disrupted by a gene trap containing a beta galactosidase (beta-gal) reporter (Eno4(+/Gt)). Expression of beta-gal occurred in the testis, and male mice homozygous for the gene trap allele (Eno4(Gt/Gt)) were infertile. Epididymal sperm numbers were 2-fold lower and sperm motility was reduced substantially in Eno4(Gt/Gt) mice compared to wild-type mice. Sperm from Eno4(Gt/Gt) mice had a coiled flagellum and a disorganized FS. The Gm5506 gene encodes a protein identical to ENO1 and also is transcribed at a low level in testis. We conclude that ENO4 is required for normal assembly of the FS and provides most of the enolase activity in sperm and that Eno1 and/or Gm5506 may encode a minor portion of the enolase activity in sperm.

Transactivating function (AF) 2-mediated AF-1 activity of estrogen receptor α is crucial to maintain male reproductive tract function.

Estrogen receptor alpha (ERα) is a ligand-dependent transcription factor containing two transcriptional activation function (AF) domains. AF-1 is in the N terminus of the receptor protein, and AF-2 activity is dependent on helix 12 of the C-terminal ligand-binding domain. We recently showed that two point mutations converting leucines 543 and 544 to alanines in helix 12 (AF2ER) minimized estrogen-dependent AF-2 transcriptional activation. A characteristic feature of AF2ER is that the estrogen antagonists ICI182780 and tamoxifen (TAM) act as agonists through intact AF-1, but not through mutated AF-2. Here we report the reproductive phenotype of male AF2ER knock-in (AF2ERKI) mice and demonstrate the involvement of ERα in male fertility. The AF2ERKI male homozygotes are infertile because of seminiferous tubular dysmorphogenesis in the testis, similar to ERα KO males. Sperm counts and motility did not differ at age 6 wk in AF2ERKI and WT mice, but a significant testis defect was observed in adult AF2ERKI male mice. The expression of efferent ductal genes involved in fluid reabsorption was significantly lower in AF2ERKI males. TAM treatment for 3 wk beginning at age 21 d activated AF-2-mutated ERα (AF2ER) and restored expression of efferent ductule genes. At the same time, the TAM treatment reversed AF2ERKI male infertility compared with the vehicle-treated group. These results indicate that the ERα AF-2 mutation results in male infertility, suggesting that the AF-1 is regulated in an AF-2-dependent manner in the male reproductive tract. Activation of ERα AF-1 is capable of rescuing AF2ERKI male infertility.

Rhox13 is translated in premeiotic germ cells in male and female mice and is regulated by NANOS2 in the male.

Male and female germ cells enter meiosis in response to an extrinsic cue by retinoic acid (RA), but the pathways downstream of RA signaling that regulate early gametogenesis remain uncertain. We identified a novel reproductive homeobox gene, Rhox13, transcribed in the prenatal ovary and testis beginning on Embryonic Day (E) 13.5. Translation of RHOX13 also begins in female germ cells on E13.5 but is suppressed in male germ cells until Postnatal Day 3. Translation of RHOX13 coincides with initiation of RA signaling in both male and female gonads in vivo but occurs precociously in neonatal testes exposed to RA in vitro or in fetal male germ cells when NANOS2 is absent in vivo. Conversely, RHOX13 translation in female germ cells is suppressed in the presence of ectopically induced NANOS2. These results strongly suggest that RHOX13 expression is regulated at a posttranscriptional step by direct interaction of NANOS2 with Rhox13 mRNA to suppress translation.

Impaired spermatogenesis and fertility in mice carrying a mutation in the Spink2 gene expressed predominantly in testes.

Spermatogenesis is a complex process involving an intrinsic genetic program composed of germ cell-specific and -predominant genes. In this study, we investigated the mouse Spink2 (serine protease inhibitor Kazal-type 2) gene, which belongs to the SPINK family of proteins characterized by the presence of a Kazal-type serine protease inhibitor-pancreatic secretory trypsin inhibitor domain. We showed that recombinant mouse SPINK2 has trypsin-inhibitory activity. Distribution analyses revealed that Spink2 is transcribed strongly in the testis and weakly in the epididymis, but is not detected in other mouse tissues. Expression of Spink2 is specific to germ cells in the testis and is first evident at the pachytene spermatocyte stage. Immunoblot analyses demonstrated that SPINK2 protein is present in male germ cells at all developmental stages, including in testicular spermatogenic cells, testicular sperm, and mature sperm. To elucidate the functional role of SPINK2 in vivo, we generated mutant mice with diminished levels of SPINK2 using a gene trap mutagenesis approach. Mutant male mice exhibit significantly impaired fertility; further phenotypic analyses revealed that testicular integrity is disrupted, resulting in a reduction in sperm number. Moreover, we found that testes from mutant mice exhibit abnormal spermatogenesis and germ cell apoptosis accompanied by elevated serine protease activity. Our studies thus provide the first demonstration that SPINK2 is required for maintaining normal spermatogenesis and potentially regulates serine protease-mediated apoptosis in male germ cells.

Lactate dehydrogenase C and energy metabolism in mouse sperm.

We demonstrated previously that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) led to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD(+) cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC.