The contributions of Dr. Nettie Stevens to the field of sex chromosome biology

The early 1900s delivered many foundational discoveries in genetics, including re-discovery of Mendel’s research and the chromosomal theory of inheritance. Following these insights, many focused their research on whether the development of separate sexes had a chromosomal basis or if instead it was caused by environmental factors. It is Dr. Nettie M. Stevens’ Studies in Spermatogenesis (1905) that provided the unequivocal evidence that the inheritance of the Y chromosome initiated male development in mealworms. This result established that sex is indeed a Mendelian trait with a genetic basis, and that the sex chromosomes play a critical role. In part II of Studies in Spermatogenesis (1906) an XY pair was identified in dozens of additional species, further validating the function of sex chromosomes. Since this formative work, a wealth of studies in animals and plants have examined the genetic basis of sex. The goal of this review is to shine a light again on Stevens’ Studies in Spermatogenesis and the lasting impact of this work. We additionally focus on key findings in plant systems over the last century and open questions that are best answered, as in Stevens’ work, by synthesizing across many systems.

Transcriptional Regulation of the Y-Linked Mammalian Testis-Determining Gene SRY

Mammalian male sex differentiation is triggered during embryogenesis by the activation of the Y-linked testis-determining gene <i>SRY</i>. Since insufficient or delayed expression of <i>SRY</i> results in XY gonadal sex reversal, accurate regulation of <i>SRY</i> is critical for male development in XY animals. In humans, dysregulation of <i>SRY</i> may cause disorders of sex development. Mouse <i>Sry</i> is the most intensively studied mammalian model of sex determination. <i>Sry</i> expression is controlled in a spatially and temporally stringent manner. Several transcription factors play a key role in sex determination as trans-acting factors for <i>Sry</i> expression. In addition, recent studies have shown that several epigenetic modifications of <i>Sry</i> are involved in sex determination as cis-acting factors for <i>Sry</i> expression. Herein, we review the current understanding of transcription factor- and epigenetic modifier-mediated regulation of <i>SRY</i>/<i>Sry</i> expression.

Female developmental environment delays development of male honeybee (Apis mellifera)

Background Nutrition and cell size play an important role in the determination of caste differentiation in queen and worker of honeybees (Apis mellifera), whereas the haploid genome dominates the differentiation of drones. However, the effects of female developmental environment on the development of males remain unclear. In this study, young drone larvae were transferred into worker cells (WCs) or remained in drone cells (DCs) to rear drones. The drone larvae were also grafted into queen cells (QCs) for 48 h and then transplanted into drone cells until emerging. Morphological indexes and reproductive organs of these three types of newly emerged drones were measured. Newly emerged drones and third instar drone larvae from WCs, DCs and QCs were sequenced by RNA sequencing (RNA-Seq). Results The amount of food remaining in cells of the QC and WC groups was significantly different to that in the DC group at the early larval stage. Morphological results showed that newly emerged DC drones had bigger body sizes and more well-developed reproductive tissues than WC and QC drones, whereas the reproductive tissues of QC drones were larger than those of WC drones. Additionally, whole body gene expression results showed a clear difference among three groups. At larval stage there were 889, 1761 and 1927 significantly differentially expressed genes (DEGs) in WC/DC, QC/DC and WC/QC comparisons, respectively. The number of DEGs decreased in adult drones of these three comparisons [678 (WC/DC), 338 (QC/DC) and 518 (WC/QC)]. A high number of DEGs were involved in sex differentiation, growth, olfaction, vision, mammalian target of rapamycin (mTOR), Wnt signaling pathways, and other processes. Conclusions This study demonstrated that the developmental environment of honeybee females can delay male development, which may serve as a model for understanding the regulation of sex differentiation and male development in social insects by environmental factors.

Effects of Functional Depletion of Doublesex on Male Development in the Sawfly, Athalia rosae

The doublesex (dsx) gene, which encodes a transcription factor, regulates sexual differentiation in insects. Sex-specific splicing of dsx occurs to yield male- and female-specific isoforms, which promote male and female development, respectively. Thus, functional disruption of dsx leads to an intersexual phenotype in both sexes. We previously identified a dsx ortholog in the sawfly, Athalia rosae. Similar to dsx in other insects, dsx in the sawfly yields different isoforms in males and females as a result of alternative splicing. The sawfly exploits a haplodiploid mode of reproduction, in which fertilized eggs develop into diploid females, whereas unfertilized eggs parthenogenetically develop into haploid males. In the present study, we knocked down the A. rosae ortholog of dsx (Ardsx) during several developmental stages with repeated double-stranded RNA (dsRNA) injections. Knockdown of Ardsx via parental RNA interference (RNAi), which enables knockdown of genes in offspring embryos, led to a lack of internal and external genitalia in haploid male progeny. Additional injection of dsRNA targeting Ardsx in these animals caused almost complete male-to-female sex reversal, but the resulting eggs were infertile. Notably, the same knockdown approach using diploid males obtained by sib-crossing caused complete male-to-female sex reversal; they were morphologically and behaviorally females. The same RNAi treatment did not affect female differentiation. These results indicate that dsx in the sawfly is essential for male development and its depletion caused complete male-to-female sex reversal. This is the first demonstration of functional depletion of dsx not causing intersexuality but inducing total sex reversal in males instead.

Plasmodium falciparum Pf77 and male development gene 1 as vaccine antigens that induce potent transmission-reducing antibodies

Malaria vaccines that disrupt the Plasmodium life cycle in mosquitoes and reduce parasite transmission in endemic areas are termed transmission-blocking vaccines (TBVs). Despite decades of research, there are only a few Plasmodium falciparum antigens that indisputably and reproducibly demonstrate transmission-blocking immunity. So far, only two TBV candidates have advanced to phase 1/2 clinical testing with limited success. By applying an unbiased transcriptomics-based approach, we have identified Pf77 and male development gene 1 (PfMDV-1) as two P. falciparum TBV antigens that, upon immunization, induced antibodies that caused reductions in oocyst counts in Anopheles mosquito midguts in a standard membrane feeding assay. In-depth studies were performed to characterize the genetic diversity of, stage-specific expression by, and natural immunity to these two molecules to evaluate their suitability as TBV candidates. Pf77 and PfMDV-1 display limited antigenic polymorphism, are pan-developmentally expressed within the parasite, and induce naturally occurring antibodies in Ghanaian adults, which raises the prospect of natural boosting of vaccine-induced immune response in endemic regions. Together, these biological properties suggest that Pf77 and PfMDV-1 may warrant further investigation as TBV candidates.

LBD Dimerization of the Androgen Receptor but Not N/C Interaction Is Crucial for Normal Male Development in Mice

The androgen receptor (AR) is a nuclear receptor with a main role in the development and maintenance of the male phenotype. To execute its role as transcription factor, the AR forms homodimers. Three dimerization modes have been described for the AR: one via the DNA binding domain, a second via the ligand binding domain (LBD) and a third via interactions between the LBD and the aminoterminus of the AR (N/C). Based exclusively on in vitro data, all three dimerization modes seem to contribute to full AR activity, albeit to a different extent. The in vivo role of the dimerization modes, however, remains unknown. To study the physiological relevance, we generated two mouse models using a CRISPR/Cas9 approach, in which either the N/C interaction (ARNoC) or LBD dimerization (ARLmon) was disrupted. Surprisingly, the male ARNoC mice have a normal phenotype, indicating that the N/C interaction is not crucial for male development. In contrast, ARLmon males have an external female phenotype with cryptorchid testes and high levels of circulating testosterone (T), androstenedione and luteinizing hormone (LH) (6-, 13- and 45-fold higher, respectively). They have no prostate, seminal vesicles or epididymis, illustrating the importance of LBD dimerization during male development. Phenotyping the ARLmon model furthermore provided evidence of a crucial role for the AR in bone homeostasis as well as steroidogenesis. The ARLmon males display a severe bone phenotype, similar to that of complete AR knockout (ARKO) mice. The bone phenotype of ARKO was postulated to be mainly due to lower estrogen levels. However, in contrast to ARKO mice, ARLmon mice have high circulating levels of T, which can still function as prohormone for estradiol and support bone function via the ERα. Immunohistological analysis of ARLmon testes showed hyperplasia of the Leydig cells and residual spermatogenesis. Analysis of the steroidogenic pathway revealed that while the expression of most genes is increased, the expression of Hsd17b3, encoding the enzyme responsible for conversion of androstenedione into T, is low in ARLmon testis. Reporter assays confirmed that the promotor of this gene is indeed upregulated by the AR itself. In conclusion, our work uncovers the physiological role of the N/C interaction and LBD dimerization of the AR. It furthermore demonstrates a direct role for AR in male bone development independent of T aromatization into estrogens. Finally, we show that the AR controls the final step in the synthesis of its own ligand. In contrast to the in vitro data, N/C interaction is not crucial for male development in vivo. The ARLmon model illustrates that LBD dimerization could be an excellent new therapeutic target for inhibiting AR activity for example in advanced prostate cancer that has developed resistance to the current AR-targeting therapies.

Transcriptional profiles in Strongyloides stercoralis males reveal deviations from the Caenorhabditis sex determination model

The human and canine parasitic nematode Strongyloides stercoralis utilizes an XX/XO sex determination system, with parasitic females reproducing by mitotic parthenogenesis and free-living males and females reproducing sexually. However, the genes controlling S. stercoralis sex determination and male development are unknown. We observed precocious development of rhabditiform males in permissive hosts treated with corticosteroids, suggesting that steroid hormones can regulate male development. To examine differences in transcript abundance between free-living adult males and other developmental stages, we utilized RNA-Seq. We found two clusters of S. stercoralis-specific genes encoding predicted transmembrane proteins that are only expressed in free-living males. We additionally identified homologs of several genes important for sex determination in Caenorhabditis species, including mab-3, tra-1, fem-2, and sex-1, which may have similar functions. However, we identified three paralogs of gld-1; Ss-qki-1 transcripts were highly abundant in adult males, while Ss-qki-2 and Ss-qki-3 transcripts were highly abundant in adult females. We also identified paralogs of pumilio domain-containing proteins with sex-specific transcripts. Intriguingly, her-1 appears to have been lost in several parasite lineages, and we were unable to identify homologs of tra-2 outside of Caenorhabditis species. Together, our data suggest that different mechanisms control male development in S. stercoralis and Caenorhabditis species.

RNA Sequencing Reveals Rice Genes Involved in Male Reproductive Development under Temperature Alteration

Rice (Oryza sativa L.) is one of the most important food crops, providing food for nearly half of the world population. Rice grain yields are affected by temperature changes. Temperature stresses, both low and high, affect male reproductive development, resulting in yield reduction. Thermosensitive genic male sterility (TGMS) rice is sterile at high temperature and fertile at low temperature conditions, facilitating hybrid production, and is a good model to study effects of temperatures on male development. Semithin sections of the anthers of a TGMS rice line under low (fertile) and high (sterile) temperature conditions showed differences starting from the dyad stage, suggesting that genes involved in male development play a role during postmeiotic microspore development. Using RNA sequencing (RNA-Seq), transcriptional profiling of TGMS rice panicles at the dyad stage revealed 232 genes showing differential expression (DEGs) in a sterile, compared to a fertile, condition. Using qRT-PCR to study expression of 20 selected DEGs using panicles of TGMS and wild type rice plants grown under low and high temperature conditions, revealed that six out of the 20 selected genes may be unique to TGMS, while the other 14 genes showed common responses to temperatures in both TGMS and wild-type rice plants. The results presented here would be useful for further investigation into molecular mechanisms controlling TGMS and rice responses to temperature alteration.

The mouse Sry locus harbors a cryptic exon that is essential for male sex determination

The mammalian sex-determining gene Sry induces male development. Since its discovery 30 years ago, Sry has been believed to be a single-exon gene. Here, we identified a cryptic second exon of mouse Sry and a corresponding two-exon type Sry (Sry-T) transcript. XY mice lacking Sry-T were sex-reversed, and ectopic expression of Sry-T in XX mice induced male development. Sry-T messenger RNA is expressed similarly to that of canonical single-exon type Sry (Sry-S), but SRY-T protein is expressed predominantly because of the absence of a degron in the C terminus of SRY-S. Sry exon2 appears to have evolved recently in mice through acquisition of a retrotransposon-derived coding sequence to replace the degron. Our findings suggest that in nature, SRY-T, not SRY-S, is the bona fide testis-determining factor.