Sexual Development

Data are from a sub-sample of black and white men and women who participate in the community- based Coronary Artery Risk Development in Young Adults (CARDIA) Study (baseline 1985) and were examined (2010) at a mean age of 50 years. The CARDIA Study [13 (link)] is a longitudinal study of the development and determinants of cardiovascular disease in 5,115 young adults aged 18–30 years at baseline in 1985–1986. The community based sample was recruited from four US cities (Birmingham, Alabama; Chicago, Illinois; Minneapolis, Minnesota; and Oakland, California) to be approximately balanced within center by sex, age (18–24 years and 25–30 years), race (white, black), and education (≤high school, >high school) [13 (link)]. In 2010 ― 2011, 72% of the surviving cohort attended the 25 year (Y25) follow-up exam. All participants provided written informed consent at each exam, and institutional review boards from each field center and the coordinating center (The University of Alabama Birmingham Institutional Review Board, University of Minnesota Institutional Review Board, Kaiser Permanente Northern California Institutional Review Board), annually approve this study.
As a part of the Y25 exam, a sub-sample of the cohort participated in the CARDIA Brain MRI sub-study. This sub-study was designed to characterize the morphology, pathology, physiology and function of the brain with magnetic resonance imaging (MRI) technology. The sample for the CARDIA Brain MRI sub-study was enrolled at the time Y25 appointments were made, with the aim of achieving a balance within four strata of ethnicity/race (black, white) and sex from three of the CARDIA field centers: Birmingham, AL, Minneapolis, MN, and Oakland, CA. Each center had a Brain MRI s target sample size and when reached enrollment was ended. Exclusion criteria at the time of sample selection, or at the MRI site, were a contra-indication to MRI or a body size that was too large for the MRI tube bore.
Separate written consent for participation in the Brain MRI sub-study was obtained, and separate approval was given by the IRBs governing participating sites (The University of Alabama Birmingham Institutional Review Board, University of Minnesota Institutional Review Board, Kaiser Permanente Northern California Institutional Review Board, University of Pennsylvania Institutional Review Board, and the NIH Office of Human Subjects Research Protection for the Intramural Research Program, National Institute on Aging).

Trichoderma reesei (H. jecorina) QM6a wild‐type strain (ATCC 13631) was used as the parental strain to construct deletions of Δvel1. CBS999.97 MAT1‐1 and MAT1‐2 strains and several other wild‐type and mutant strains from different sources were included in this study to set up informative crosses (Table 1). Female fertile strains FF1 and FF2 with QM6a background were prepared as described previously (Schuster et al., 2012), but with 10 instead of 5 crosses.
Propagation of strains was performed on 3% (w/v) malt extract agar (Merck, Darmstadt, Germany). Crossing experiments were made on 2% (w/v) malt extract agar at 22°C in daylight (cycles of 12 h light–12 h dark) or constant darkness. Therefore, strains were grown on opposite sides of petri dishes and were evaluated for fruiting body formation and ascospore discharge 7 and 20 days after inoculation respectively. For evaluation of conidiation in QM6a Δvel1, Mandels–Andreotti (MA) medium (Mandels and Andreotti, 1978) supplemented with 1% (w/v) carbon source and 0.1% (w/v) peptone (Merck) to induce germination was used. D‐galactose (Sigma Aldrich, St. Louis, USA), D‐sorbitol (ALFA AESAR, Karlsruhe, Germany), D‐mannitol (Fluka, St. Gallen, Switzerland), D‐arabinose (Sigma Aldrich), meso‐erythritol (ALFA AESAR) and γ‐amino butyric acid (GABA) (Sigma Aldrich) or D(+)‐xylose (Sigma Aldrich) were used as carbon sources in sporulation assays.
For transcriptional analysis, the strains were pre‐cultured on 3% (w/v) malt extract agar in constant darkness for at least 3 days. Inoculation of strains was performed on 2% (w/v) malt extract agar plates covered with cellophane to facilitate harvesting. To study gene expression in the course of sexual development, the strains were inoculated on both sides of the petri dishes and the mycelia of the partner strains were harvested at the stage of contact (3 days after inoculation). As the mutant strains have growth defects compared with wild‐type strains, they were inoculated in different distances from the opposite mating strain on petri dishes in order to obtain the contact stage of mycelia for the mutant strains at consistent times. Five plates at the stage of contact were pooled and at least two biological replicates were used for quantitative reverse transcription polymerase chain reaction (qRT‐PCR) analysis. In order to compare the obtained data with asexual growth, strains were grown alone on plates and the mycelia were harvested at the time points corresponding to the contact of strains in sexual development. The strains were grown under cycles of 12 h light–12 h darkness at 22°C (1800 lux).
For cellulase screening, strains were grown on plates with MA medium containing 1% (w/v) carboxymethylcellulose (CMC) (Sigma Aldrich) and after 2 days were stained with Congo red (0.1% w/v solution in water) (Roth, Karlsruhe, Germany), which stains cellulose (Carder, 1986). After washing of the plate with 1 M NaCl, halos indicate cellulase production. Addition of 1% (w/v) lactose or glucose (both from Sigma Aldrich) was used as additional controls. Escherichia coli JM109 was used for cloning (Yanisch‐Perron et al., 1985).

To test for the presence of modules in the gene regulatory network of sexual development, we employed the WGCNA R package (v1.69) (Langfelder & Horvath, 2008 (link); Zhang & Horvath, 2005 (link)) to construct (i) a turtle consensus network, (ii) a Chrysemys‐specific network, and (iii) an Apalone‐specific network from reciprocal best blast hits between the two transcriptomes. To construct the modules, we followed guidance provided by the online tutorials (https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/Tutorials/). Counts were rounded to integers, filtered to a minimum cross‐library read count of at least 20, and transformed using varianceStabilizingTransformation() from the DESeq2 package. Data were then cleaned and clustered, and a soft‐power of 8 was selected as it best met the assumption of a scale‐free topology. Then, consensus and species modules were built.
Weighted gene correlation network analysis uses (1) correlation to measure co‐expression and thus, interaction among genes; (2) hierarchical clustering to identify co‐expression modules (highly correlated groups of genes); and (3) eigengene network analysis to define module relationships (Langfelder & Horvath, 2008 (link)). Briefly, network nodes are gene expression profiles, edges between genes are the pairwise correlations between their gene expression, and connectivity is how highly co‐expressed a gene is relative to other genes in a module (Langfelder & Horvath, 2008 (link)). A module's eigengene is its principal component, or representative (weighted average) gene expression profile. When interpreting gene co‐expression data, we are mindful that these species‐specific networks are based on highly heterogeneous data that vary by sex, temperature, and embryonic stage/tissue, which could influence the construction of networks.
Overlap between consensus and species‐specific modules (and the genes involved) were calculated, as well as the network adjacency (i.e., connection strength between nodes) and preservation (i.e., conservation or similarity) among species‐specific modules. Given the high conservation of the vertebrate sex determination network (Merchant‐Larios et al., 2021 (link); Morrish & Sinclair, 2002 (link)), we predicted that high module overlap would exist, but also that some differences in the molecular circuitry of TSD and GSD mechanism would be present between Chrysemys and Apalone.

The parental questionnaire was developed based on a series of published studies (Chen and Chen, 2005 (link); Dake et al., 2014 (link); Morawska et al., 2015 (link); Robinson et al., 2017 (link); Depauli and Plaute, 2018 (link); Rudolph and Zimmer-Gembeck, 2018 (link); UNESCO, 2018 ; Shin et al., 2019 (link)) and included 15 items on knowledge, attitudes, and practices regarding sexuality education of children.
The knowledge subscale included five items, including knowledge of correct terminology for genitalia, daily sexual healthcare, child sexual development, and sexuality questions, that may be encountered at different ages of development of children, and accurate information on child sexual abuse prevention education, “e.g., that do you know the correct terminology for genitalia”. Response options were “yes,” “no,” or “unsure” for each item. “Yes” responses scored 1, while “no” and unsure responses scored 0. The scores for each item were summed for a total knowledge score (range = 0–5). A brief attitudes subscale consists of three items that ask whether parents agree or disagree with the aspects of sexuality education for children in primary school (0–3). The practices subscale included seven items. These seven questions were asked about parental communication with their child about sexuality. Examples of questions in this section included the following: “Did you use appropriate terminology with your child in the process of sexuality education?” “Did you encourage your child to share their thoughts and feelings about sexuality?” and “Did you give brochures or other materials to your child to help them learn about sexuality?” Response options were “yes,” “no,” or “unsure” for each item. “Yes” responses scored 1, while “no” and unsure responses scored 0. The scores for each item were summed for a total practice score (range = 0–7). Internal consistency analyses of subscales of knowledge, attitudes, and practices produced alpha levels of 0.83, 0.90, and 0.80, respectively.