Male animal sterilization: history, current practices, and potential methods for replacing castration.

Sterilization and castration have been synonyms for thousands of years. Making an animal sterile meant to render them incapable of producing offspring. Castration or the physical removal of the testes was discovered to be the most simple but reliable method for managing reproduction and sexual behavior in the male. Today, there continues to be global utilization of castration in domestic animals. More than six hundred million pigs are castrated every year, and surgical removal of testes in dogs and cats is a routine practice in veterinary medicine. However, modern biological research has extended the meaning of sterilization to include methods that spare testis removal and involve a variety of options, from chemical castration and immunocastration to various methods of vasectomy. This review begins with the history of sterilization, showing a direct link between its practice in man and animals. Then, it traces the evolution of concepts for inducing sterility, where research has overlapped with basic studies of reproductive hormones and the discovery of testicular toxicants, some of which serve as sterilizing agents in rodent pests. Finally, the most recent efforts to use the immune system and gene editing to block hormonal stimulation of testis function are discussed. As we respond to the crisis of animal overpopulation and strive for better animal welfare, these novel methods provide optimism for replacing surgical castration in some species.

Cryptorchidism and testicular cancer in the dog: unresolved questions and challenges in translating insights from human studies†.

Cryptorchidism, the failure of one or both testes to descend into the scrotum, and testicular cancer show a strong correlation in both dogs and humans. Yet, long-standing medical debates persist about whether the location of undescended testes directly causes testicular cancer in humans or if both conditions stem from a common origin. Although testicular cancer is a prevalent disease in dogs, even less is known about its cause and correlation with testicular descent in this species. This review investigates the relation between these two disorders in dogs, drawing insights from human studies, and examines key biomarkers identified thus far. In addition, it explores potential causal links, including the impact of temperature on maturing testicular cells and a potential shared genetic origin. Notably, this literature review reveals significant differences between men and dogs in reproductive development, histological and molecular features of testicular tumors, and the prevalence of specific tumor types, such as Sertoli cell tumors in cryptorchid dogs and germ cell tumors in humans. These disparities caution against using dogs as models for human testicular cancer research and underscore the limitations when drawing comparisons between species. The paper concludes by suggesting specific research initiatives to enhance our understanding of the complex interplay between cryptorchidism and testicular cancer in dogs.

Role for Nongenomic Estrogen Signaling in Male Fertility.

Estrogen actions are mediated by both nuclear (n) and membrane (m) localized estrogen receptor 1 (ESR1). Male Esr1 knockout (Esr1KO) mice lacking functional Esr1 are infertile, with reproductive tract abnormalities. Male mice expressing nESR1 but lacking mESR1 (nuclear-only estrogen receptor 1 mice) are progressively infertile due to testicular, rete testis, and efferent ductule abnormalities similar to Esr1KO males, indicating a role for mESR1 in male reproduction. The H2NES mouse expresses only mESR1 but lacks nESR1. The goal of this study was to identify the functions of mESR1 alone in mice where nESR1 was absent. Breeding trials showed that H2NES males are fertile, with decreased litter numbers but normal pup numbers/litter. In contrast to Esr1KO mice, H2NES testicular, and epididymal weights were not reduced, and seminiferous tubule abnormalities were less pronounced. However, Esr1KO and H2NES males both had decreased sperm motility and a high incidence of abnormal sperm morphology. Seminiferous tubule and rete testis dilation and decreased efferent ductule epithelial height characteristic of Esr1KO males were reduced in H2NES. Consistent with this, expression of genes involved in fluid transport and ion movement that were reduced in Esr1KO (Aqp1, Car2, Car14, Cftr) were partially or fully restored to wild-type levels in H2NES. In summary, in contrast to Esr1KO males, H2NES males are fertile and have reduced phenotypic and functional abnormalities in the testis and efferent ductules. Thus, mESR1 alone, in the absence of nESR1, can partially regulate male reproductive tract structure and function, emphasizing its importance for overall estrogen action.

MIWI N-terminal RG motif promotes efficient pachytene piRNA production and spermatogenesis independent of LINE1 transposon silencing.

PIWI proteins and their associated piRNAs act to silence transposons and promote gametogenesis. Murine PIWI proteins MIWI, MILI, and MIWI2 have multiple arginine and glycine (RG)-rich motifs at their N-terminal domains. Despite being known as docking sites for the TDRD family proteins, the in vivo regulatory roles for these RG motifs in directing PIWI in piRNA biogenesis and spermatogenesis remain elusive. To investigate the functional significance of RG motifs in mammalian PIWI proteins in vivo, we genetically engineered an arginine to lysine (RK) point mutation of a conserved N-terminal RG motif in MIWI in mice. We show that this tiny MIWI RG motif is indispensable for piRNA biogenesis and male fertility. The RK mutation in the RG motif disrupts MIWI-TDRKH interaction and impairs enrichment of MIWI to the intermitochondrial cement (IMC) for efficient piRNA production. Despite significant overall piRNA level reduction, piRNA trimming and maturation are not affected by the RK mutation. Consequently, MiwiRK mutant mice show chromatoid body malformation, spermatogenic arrest, and male sterility. Surprisingly, LINE1 transposons are effectively silenced in MiwiRK mutant mice, indicating a LINE1-independent cause of germ cell arrest distinctive from Miwi knockout mice. These findings reveal a crucial function of the RG motif in directing PIWI proteins to engage in efficient piRNA production critical for germ cell progression and highlight the functional importance of the PIWI N-terminal motifs in regulating male fertility.

SYPL1 defines a vesicular pathway essential for sperm cytoplasmic droplet formation and male fertility.

The cytoplasmic droplet is a conserved dilated area of cytoplasm situated at the neck of the sperm flagellum. Viewed as residual cytoplasm inherited from late spermatids, the cytoplasmic droplet contains numerous saccular elements as its key content. However, the origin of these saccules and the function of the cytoplasmic droplet have long been speculative. Here, we identify the molecular origin of these cytoplasmic droplet components by uncovering a vesicle pathway essential for formation and sequestration of saccules within the cytoplasmic droplet. This process is governed by a transmembrane protein SYPL1 and its interaction with VAMP3. Genetic ablation of SYPL1 in mice reveals that SYPL1 dictates the formation and accumulation of saccular elements in the forming cytoplasmic droplet. Derived from the Golgi, SYPL1 vesicles are critical for segregation of key metabolic enzymes within the forming cytoplasmic droplet of late spermatids and epididymal sperm, which are required for sperm development and male fertility. Our results uncover a mechanism to actively form and segregate saccules within the cytoplasmic droplet to promote sperm fertility.

Single neonatal estrogen implant sterilizes female animals by decreasing hypothalamic KISS1 expression.

Reproductive sterilization by surgical gonadectomy is strongly advocated to help manage animal populations, especially domesticated pets, and to prevent reproductive behaviors and diseases. This study explored the use of a single-injection method to induce sterility in female animals as an alternative to surgical ovariohysterectomy. The idea was based on our recent finding that repetitive daily injection of estrogen into neonatal rats disrupted hypothalamic expression of Kisspeptin (KISS1), the neuropeptide that triggers and regulates pulsatile secretion of GnRH. Neonatal female rats were dosed with estradiol benzoate (EB) either by daily injections for 11 days or by subcutaneous implantation of an EB-containing silicone capsule designed to release EB over 2-3 weeks. Rats treated by either method did not exhibit estrous cyclicity, were anovulatory, and became infertile. The EB-treated rats had fewer hypothalamic Kisspeptin neurons, but the GnRH-LH axis remained responsive to Kisspeptin stimulation. Because it would be desirable to use a biodegradable carrier that is also easier to handle, an injectable EB carrier was developed from PLGA microspheres to provide pharmacokinetics comparable to the EB-containing silicone capsule. A single neonatal injection of EB-microspheres at an equivalent dosage resulted in sterility in the female rat. In neonatal female Beagle dogs, implantation of an EB-containing silicone capsule also reduced ovarian follicle development and significantly inhibited KISS1 expression in the hypothalamus. None of the treatments produced any concerning health effects, other than infertility. Therefore, further development of this technology for sterilization in domestic female animals, such as dogs and cats is worthy of investigation.

Expression patterns of sex steroid receptors in developing mesonephros of the male mouse: three-dimensional analysis.

The androgen pathway via androgen receptor (AR) has received the most attention for development of male reproductive tracts. The estrogen pathway through estrogen receptor (ESR1) is also a major contributor to rete testis and efferent duct formation, but the role of progesterone via progesterone receptor (PGR) has largely been overlooked. Expression patterns of these receptors in the mesonephric tubules (MTs) and Wolffian duct (WD), which differentiate into the efferent ductules and epididymis, respectively, remain unclear because of the difficulty in distinguishing each region of the tracts. This study investigated AR, ESR1, and PGR expressions in the murine mesonephros using three-dimensional (3-D) reconstruction. The receptors were localized in serial paraffin sections of the mouse testis and mesonephros by immunohistochemistry on embryonic days (E) 12.5, 15.5, and 18.5. Specific regions of the developing MTs and WD were determined by 3-D reconstruction using Amira software. AR was found first in the specific portion of the MTs near the MT-rete junction at E12.5, and the epithelial expression showed increasing strength from cranial to the caudal regions. Epithelial expression of ESR1 was found in the cranial WD and MTs near the WD first at E15.5. PGR was weakly positive only in the MTs and cranial WD starting on E15.5. This 3-D analysis suggests that gonadal androgen acts first on the MTs near the MT-rete junction but that estrogen is the first to influence MTs near the WD, while potential PGR activity is delayed and limited to the epithelium.

DNALI1 interacts with the MEIG1/PACRG complex within the manchette and is required for proper sperm flagellum assembly in mice.

The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii, recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.

Disrupted intercellular bridges and spermatogenesis in fatty acyl-CoA reductase 1 knockout mice: A new model of ether lipid deficiency.

Peroxisomal fatty acyl-CoA reductase 1 (FAR1) is a rate-limiting enzyme for ether lipid (EL) synthesis. Gene mutations in FAR1 cause a rare human disease. Furthermore, altered EL homeostasis has also been associated with various prevalent human diseases. Despite their importance in human health, the exact cellular functions of FAR1 and EL are not well-understood. Here, we report the generation and initial characterization of the first Far1 knockout (KO) mouse model. Far1 KO mice were subviable and displayed growth retardation. The adult KO male mice had smaller testes and were infertile. H&E and immunofluorescent staining showed fewer germ cells in seminiferous tubules. Round spermatids were present but no elongated spermatids or spermatozoa were observed, suggesting a spermatogenesis arrest at this stage. Large multi-nucleated giant cells (MGC) were found lining the lumen of seminiferous tubules with many of them undergoing apoptosis. The immunofluorescent signal of TEX14, an essential component of intercellular bridges (ICB) between developing germ cells, was greatly reduced and mislocalized in KO testis, suggesting the disrupted ICBs as an underlying cause of MGC formation. Integrative analysis of our total testis RNA-sequencing results and published single-cell RNA-sequencing data unveiled cell type-specific molecular alterations underlying the spermatogenesis arrest. Many genes essential for late germ cell development showed dramatic downregulation, whereas genes essential for extracellular matrix dynamics and cell-cell interactions were among the most upregulated genes. Together, this work identified the cell type-specific requirement of ELs in spermatogenesis and suggested a critical role of Far1/ELs in the formation/maintenance of ICB during meiosis.

A single amino acid mutation in the mouse MEIG1 protein disrupts a cargo transport system necessary for sperm formation.

Mammalian spermatogenesis is a highly coordinated process that requires cooperation between specific proteins to coordinate diverse biological functions. For example, mouse Parkin coregulated gene (PACRG) recruits meiosis-expressed gene 1 (MEIG1) to the manchette during normal spermiogenesis. Here we mutated Y68 of MEIG1 using the CRISPR/cas9 system and examined the biological and physiological consequences in mice. All homozygous mutant males examined were completely infertile, and sperm count was dramatically reduced. The few developed sperm were immotile and displayed multiple abnormalities. Histological staining showed impaired spermiogenesis in these mutant mice. Immunofluorescent staining further revealed that this mutant MEIG1 was still present in the cell body of spermatocytes, but also that more MEIG1 accumulated in the acrosome region of round spermatids. The mutant MEIG1 and a cargo protein of the MEIG1/PACRG complex, sperm-associated antigen 16L (SPAG16L), were no longer found to be present in the manchette; however, localization of the PACRG component was not changed in the mutants. These findings demonstrate that Y68 of MEIG1 is a key amino acid required for PACRG to recruit MEIG1 to the manchette to transport cargo proteins during sperm flagella formation. Given that MEIG1 and PACRG are conserved in humans, small molecules that block MEIG1/PACRG interaction are likely ideal targets for the development of male contraconception drugs.

Insights into differentiation and function of the transition region between the seminiferous tubule and rete testis.

Seminiferous tubules physically connect to the rete testis through short segments called the transition region (TR). During fetal development, this specialized junction is considered the initial site where testis cords begin to form and to grow in length well beyond birth and into adulthood and form convoluted tubular cores. Mitotic activity of the Sertoli cell, the somatic cell of the epithelium, ceases before puberty, but modified Sertoli cells in the TR remain immature and capable of proliferation. This review presents what is known about this specialized region of the testis, with an emphasis on the morphological, molecular and physiological features, which support the hypothesis that this short region of epithelial transition serves as a specialized niche for undifferentiated Sertoli cells and spermatogonial stem cells. Also, the region is populated by an elevated number of immune cells, suggesting an important activity in monitoring and responding to any leakage of autoantigens, as sperm enter the rete testis. Several structure/function characteristics of the transition region are discussed and compared across species.

CEP164 is essential for efferent duct multiciliogenesis and male fertility.

Cilia are evolutionarily conserved microtubule-based structures that perform diverse biological functions. Cilia are assembled on basal bodies and anchored to the plasma membrane via distal appendages. In the male reproductive tract, multicilia in efferent ducts (EDs) move in a whip-like motion to prevent sperm agglutination. Previously, we demonstrated that the distal appendage protein CEP164 recruits Chibby1 (Cby1) to basal bodies to facilitate basal body docking and ciliogenesis. Mice lacking CEP164 in multiciliated cells (MCCs) (FoxJ1-Cre;CEP164fl/fl) show a significant loss of multicilia in the trachea, oviduct, and ependyma. In addition, we observed male sterility; however, the precise role of CEP164 in male fertility remained unknown. Here, we report that the seminiferous tubules and rete testis of FoxJ1-Cre;CEP164fl/fl mice exhibit substantial dilation, indicative of dysfunctional multicilia in the EDs. We found that multicilia were hardly detectable in the EDs of FoxJ1-Cre;CEP164fl/fl mice although FoxJ1-positive immature cells were present. Sperm aggregation and agglutination were commonly noticeable in the lumen of the seminiferous tubules and EDs of FoxJ1-Cre;CEP164fl/fl mice. In FoxJ1-Cre;CEP164fl/fl mice, the apical localization of Cby1 and the transition zone marker NPHP1 was severely diminished, suggesting basal body docking defects. TEM analysis of EDs further confirmed basal body accumulation in the cytoplasm of MCCs. Collectively, we conclude that male infertility in FoxJ1-Cre;CEP164fl/fl mice is caused by sperm agglutination and obstruction of EDs due to loss of multicilia. Our study, therefore, unravels an essential role of the distal appendage protein CEP164 in male fertility.

Leucine zipper transcription factor-like 1 (LZTFL1), an intraflagellar transporter protein 27 (IFT27) associated protein, is required for normal sperm function and male fertility.

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella, including mammalian sperm tails. Depletion of IFT27, a component of the IFT complex, in male germ cells results in infertility associated with disrupted sperm flagella structure and motility. Leucine zipper transcription factor-like 1 (LZTFL1) is an IFT27 associated protein. LZTFL1, also known as BBS17, is a Bardet-Biedl syndrome (BBS) associated protein. Patients carrying biallelic variants of LZTFL1 gene exhibit the common BBS phenotypes. The global Lztfl1 knockout mice showed abnormal growth rate and retinal degeneration, typical of BBS phenotype. However, it is not clear if Lztfl1 has a role in male fertility. The LZTFL1 protein is highly and predominantly expressed in mouse testis. During the first wave of spermatogenesis, the protein is only expressed during spermiogenesis phase from the round spermatid stage and displays a cytoplasmic localization with a vesicular distribution pattern. At the elongated spermatid stage, LZTFL1 is present in the developing flagella and appears also close to the manchette. Fertility of Lztfl1 knockout mice was significantly reduced and associated with low sperm motility and a high level of abnormal sperm (astheno-teratozoospermia). In vitro assessment of fertility revealed reduced fertilization and embryonic development when using sperm from homozygous mutant mice. In addition, we observed a significant decrease of the testicular IFT27 protein level in Lztfl1 mutant mice contrasting with a stable expression levels of other IFT proteins, including IFT20, IFT81, IFT88 and IFT140. Overall, our results support strongly the important role of LZTFL1 in mouse spermatogenesis and male fertility.

Estrogens and development of the rete testis, efferent ductules, epididymis and vas deferens.

Estrogen has always been considered the female hormone and testosterone the male hormone. However, estrogen's presence in the testis and deleterious effects of estrogen treatment during development have been known for nearly 90 years, long before estrogen receptors (ESRs) were discovered. Eventually it was learned that testes actually synthesize high levels of estradiol (E2) and sequester high concentrations in the reproductive tract lumen, which seems contradictory to the overwhelming number of studies showing reproductive pathology following exogenous estrogen exposures. For too long, the developmental pathology of estrogen has dominated our thinking, even resulting in the "estrogen hypothesis" as related to the testicular dysgenesis syndrome. However, these early studies and the development of an Esr1 knockout mouse led to a deluge of research into estrogen's potential role in and disruption of development and function of the male reproductive system. What is new is that estrogen action in the male cannot be divorced from that of androgen. This paper presents what is known about components of the estrogen pathway, including its synthesis and target receptors, and the need to achieve a balance between androgen- and estrogen-action in male reproductive tract differentiation and adult functions. The review focuses on what is known regarding development of the male reproductive tract, from the rete testis to the vas deferens, and examines the expression of estrogen receptors and presence of aromatase in the male reproductive system, traces the evidence provided by estrogen-associated knockout and transgenic animal models and discusses the effects of fetal and postnatal exposures to estrogens. Hopefully, there will be enough here to stimulate discussions and new investigations of the androgen:estrogen balance that seems to be essential for development of the male reproductive tract.

The protein YWHAE (14-3-3 epsilon) in spermatozoa is essential for male fertility.

BACKGROUND: Spermatogenesis is a complex biological process highlighted by synthesis and activation of proteins that regulate meiosis and cellular differentiation occur during spermatogenesis. 14-3-3 proteins are adaptor proteins that play critical roles in kinase signaling, especially for regulation of cell cycle and apoptosis in eukaryotic cells. There are seven isoforms of the 14-3-3 family proteins encoded by seven genes (ß, ε, γ, η, θ/τ, ζ and σ). 14-3-3 isoforms have been shown to have many interacting partners in several tissues including testis. OBJECTIVE: While it is known that 14-3-3 proteins are expressed in the functions of testis and spermatozoon, the role for each of the seven isoforms is not known. In this study, we investigated the roles of 14-3-3η and 14-3-3ε isoforms in spermatogenesis. MATERIALS AND METHODS: To study the in vivo function of 14-3-3η and 14-3-3ε in spermatogenesis, we generated testis-specific and global knockout mice for each of 14-3-3η and 14-3-3ε isoforms (CKO and GKO, respectively). Computer-assisted semen analysis was used to assess sperm motility, while immunohistochemical studies were conducted to check spermatogenesis. RESULTS: Although both 14-3-3η and 14-3-3ε isoforms were present in mouse testis, only the expression of 14-3-3ε, but not 14-3-3η, was detected in spermatozoa. Mice lacking 14-3-3η were normal and fertile while 14-3-3ε CKO and GKO males showed infertility. Low sperm count with higher abnormal spermatozoa was seen in 14-3-3ε CKO mice. The motility of 14-3-3ε knockout spermatozoa was lower than that of the control. A reduction in the phosphorylation of both glycogen synthase kinase 3 and PP1γ2 was also seen in spermatozoa from 14-3-3ε CKO mice, suggesting a specific role of 14-3-3ε in spermatogenesis, sperm motility, and fertility. DISCUSSION AND CONCLUSION: This is the first demonstration that of the seven 14-3-3 isoforms, 14-3-3ε is essential for normal sperm function and male fertility.

Autophagy core protein ATG5 is required for elongating spermatid development, sperm individualization and normal fertility in male mice.

Spermiogenesis is the longest phase of spermatogenesis, with dramatic morphological changes and a final step of spermiation, which involves protein degradation and the removal of excess cytoplasm; therefore, we hypothesized that macroautophagy/autophagy might be involved in the process. To test this hypothesis, we examined the function of ATG5, a core autophagy protein in male germ cell development. Floxed Atg5 and Stra8- iCre mice were crossed to conditionally inactivate Atg5 in male germ cells. In Atg5flox/flox; Stra8- iCre mutant mice, testicular expression of the autophagosome marker LC3A/B-II was significantly reduced, and expression of autophagy receptor SQSTM1/p62 was significantly increased, indicating a decrease in testicular autophagy activity. The fertility of mutant mice was dramatically reduced with about 70% being infertile. Sperm counts and motility were also significantly reduced compared to controls. Histological examination of the mutant testes revealed numerous, large residual bodies in the lumen of stages after their normal resorption within the seminiferous epithelium. The cauda epididymal lumen was filled with sloughed germ cells, large cytoplasmic bodies, and spermatozoa with disorganized heads and tails. Examination of cauda epididymal sperm by electron microscopy revealed misshapen sperm heads, a discontinuous accessory structure in the mid-piece and abnormal acrosome formation and loss of sperm individualization. Immunofluorescence staining of epididymal sperm showed abnormal mitochondria and acrosome distribution in the mutant mice. ATG5 was shown to induce autophagy by mediating multiple signals to maintain normal developmental processes. Our study demonstrated ATG5 is essential for male fertility and is involved in various aspects of spermiogenesis.Abbreviations: AKAP4: a-kinase anchoring protein 4; ATG5: autophagy-related 5; ATG7: autophagy-related 7; ATG10: autophagy-related 10; ATG12: autophagy-related 12; cKO: conditional knockout; DDX4: DEAD-box helicase 4; MAP1LC3/LC3/tg8: microtubule-associated protein 1 light chain 3; PBS: phosphate-buffered saline; PIWIL2/MILI: piwi like RNA-mediated gene silencing 2; RT-PCR: reverse transcription-polymerase chain reaction; SQSTM1/p62: sequestosome 1; TBC: tubulobulbar complexes; WT: wild type.

Hypothyroidism induced by postnatal PTU (6-n-propyl-2-thiouracil) treatment decreases Sertoli cell number and spermatogenic efficiency in sexually mature pigs.

Studies with 6-n-propyl-2-thiouracil (PTU) in laboratory rodents have shown that transient neonatal hypothyroidism leads to increased Sertoli cell (SC) number, testis size and sperm production. However, scarce and inconclusive data are available for farm animals. In the present study, Piau pigs received PTU in a gel capsule containing 8 mg/kg of body weight for 14 weeks starting from the first week of age, whereas control animals received only the vehicle. Blood samples were collected during the experimental period for hormonal evaluation in the serum. The animals were orchiectomized at adulthood and had their testes used for histomorphometric analysis. Indicating that the PTU concentration used was effective in promoting hypothyroidism, PTU-treated pigs showed a 30% lower body weight and reduced thyroxine levels (p < 0.05) during the treatment period. At adulthood, the body weight was similar in both groups but, surprisingly, PTU-treated pigs showed 30% lower testis weight (p < 0.05). In general, treated pigs presented increased follicle-stimulating hormone levels, whereas testosterone levels tended to be lower from 9 to 23 weeks of age. No significant differences were observed for estradiol, Leydig cell volume and number, tubular diameter, SC number per gram of testis, SC efficiency and meiotic index. However, seminiferous tubule occupancy, total tubular length, SC number per testis, and daily sperm production per testis and per gram of testis (DSP/g/T) were significantly lower (p < 0.05) in PTU-treated pigs. Therefore, in contrast to laboratory rodents, our results showed that SC proliferation and DSP/g/T (spermatogenic efficiency) in Piau pigs is diminished by postnatal PTU treatment.

Acrosomal marker SP-10 (gene name Acrv1) for staging of the cycle of seminiferous epithelium in the stallion.

The acrosome plays a critical role in sperm-oocyte interactions during fertilization. SP-10 is an acrosomal matrix protein, which is evolutionarily conserved among mammals. The SP-10 antibody has been shown to be useful for staging the seminiferous cycle in the mouse and human. A canonical acrosomal marker; however, has never been used for staging in the horse. The objectives of the present study were to investigate the presence of SP-10 within the horse acrosome using an anti-mouse SP-10 antibody, to classify spermatids based on the shape of the acrosome, and then to use that information to assign stages of the cycle of the seminiferous epithelium. Testes from mature stallions with history of normospermic ejaculates were used for immunohistochemistry. We found that the mouse SP-10 antibody stained the horse acrosome vividly in testis cross-sections, indicating evolutionary conservation. Previous methods based on morphology alone without the aid of an antibody marker showed 8 stages in the horse seminiferous epithelium. Morphological detail of the acrosome afforded by the SP-10 marker in this study identified 16 steps of spermatids. This, in turn, led to the identification of 12 distinct stages in the cycle of the seminiferous epithelium of the horse wherein stage I shows recently formed round spermatids and stage XII includes meiotic divisions; a classification that is consistent with other animal models. The SP-10 antibody marks the acrosome in a way that enables researchers in the field to identify stages of spermatogenesis in the horse easily. In conclusion, we demonstrated that immunolabeling for SP-10 can be an objective approach to stage the cycle of the seminiferous epithelium in normospermic stallions; future studies will determine if SP-10 could be used to assess testicular dysfunction.

The essential role of intraflagellar transport protein IFT81 in male mice spermiogenesis and fertility.

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism that is indispensable for the formation and maintenance of cilia and flagella; however, the implications and functions of IFT81 remain unknown. In this study, we disrupted IFT81 expression in male germ cells starting from the spermatocyte stage. As a result, homozygous mutant males were completely infertile and displayed abnormal sperm parameters. In addition to oligozoospermia, spermatozoa presented dysmorphic and nonfunctional flagella. Histological examination of testes from homozygous mutant mice revealed abnormal spermiogenesis associated with sloughing of germ cells and the presence of numerous multinucleated giant germ cells (symblasts) in the lumen of seminiferous tubules and epididymis. Moreover, only few elongated spermatids and spermatozoa were seen in analyzed cross sections. Transmission electron microscopy showed a complete disorganization of the axoneme and para-axonemal structures such as the mitochondrial sheath, fibrous sheath, and outer dense fibers. In addition, numerous vesicles that contain unassembled microtubules were observed within developing spermatids. Acrosome structure analysis showed normal appearance, thus excluding a crucial role of IFT81 in acrosome biogenesis. These observations showed that IFT81 is an important member of the IFT process during spermatogenesis and that its absence is associated with abnormal flagellum formation leading to male infertility. The expression levels of several IFT components in testes, including IFT20, IFT25, IFT27, IFT57, IFT74, and IFT88, but not IFT140, were significantly reduced in homozygous mutant mice. Overall, our study demonstrates that IFT81 plays an essential role during spermatogenesis by modulating the assembly and elongation of the sperm flagella.