Comparison of rat fetal sex determination using placental gDNA and mRNA via qRT-PCR

Abstract

A growing need exists to consider fetal sex as a biological variable and accurately assess sex-specific effects. Among the multiple methods used to determine fetal sex, quantitative real-time polymerase chain reaction (qRT-PCR) of Sry (sex-determining region Y) with genomic DNA (gDNA) is commonly used in addition to use of methodologies such as transcriptomics and detection of Barr body. However, Sry messenger RNA (mRNA), a product of Sry gDNA, has not been previously assessed for sex determination. Using placental samples from timed-pregnant Wistar rats at gestational day (GD) 16, this study assessed the compatibility of Sry detection using gDNA versus mRNA to determine fetal sex. Samples used in this current study come from a larger study that investigated trichloroethylene (TCE) reproductive toxicity and potential modulation by N-acetyl-L-cysteine (NAC) and aminooxyacetic acid (AOAA). In 90 out of 91 samples, the sex classification determined by gDNA matched the sex classification determined by mRNA analyzing Sry (Sry/B2m) values. For both gDNA and mRNA, statistically significant differences in Sry/B2m values between males and females were observed with samples considered in totality and when samples were separated by treatment groups (all comparisons were p<0.01 or below, and all but two comparisons were p<0.001 or below). Finally, the validity of using Sry Cq values to determine fetal sex and the B2m reference gene were also discussed. Together, this study suggests that determination of fetal sex in Wistar rats can be accomplished using Sry measurements in gDNA or mRNA with highly compatible results.

Introduction

With increased emphasis of sex as a biological variable that influences disease, behavior, and other outcomes [1], the accurate determination of fetal sex in placental samples of otherwise unknown sex is increasingly important. Multiple methods are available to determine sex of a biological sample, each with its limitations and strengths. Among the multiple methods to determine sex of a biological sample, the newest technologies provide novel opportunities and methods for sex determination. For instance, single-cell RNA sequencing has provided precision over more traditional transcriptomic approaches, including microarrays and RNA-sequencing, to allow for the determination of sex of a single cell versus a population of cells [2]. Transcriptomic approaches can provide ample information, but considerations including cost and project purpose may limit use of a transcriptomic approach to determine sex.

Traditional approaches that are less expensive than the aforementioned “-omics” technologies are available to determine sex, albeit with their own limitations and other considerations. In samples not limited to mammals, the detection of sex-specific genes, such as Sry (sex-determining region Y), can be performed using Southern blot, nested polymerase chain reaction (PCR), or quantitative PCR methods. Consideration of these approaches include execution difficulty and the retrieval of only genomic, not protein-level, information. Detection of enzymatic activities that are known to differ by sex, including activity of CYP3A4, CYP2B6, and methyltransferases [3, 4], can be used but care should be taken to establish thresholds and consider other factors that may influence enzymatic activity. Therefore, in both above-mentioned approaches, appropriate controls should be used to rule out false positive or false negative readings.

In mammalian samples, the detection of the Barr body, detection of the H-Y antigen, or consideration of fetal germ cell development (only in humans) has been used to attempt to determine the sex of the individual [5–12]. The Barr body is formed from a silenced X chromosome and is only present in female samples [5]. However, it may not always be detected in females [6, 9, 10] and its absence, therefore, could yield a false reading of sex. In contrast, the H-Y antigen is theoretically only present in males, appears as early as the eight-cell stage, and can differentiate males from females, but suffers from a complex detection method and may not be sufficiently accurate because false positive and negative findings have been reported [7, 11, 12]. Assessment of events in fetal germ cell development in humans can be used to determine sex. As an example, retinoic acid signaling is specific to females [8]. The timing of events in fetal germ cell development can also be assessed to determine sex. An example of this is the case of mitosis, which happens in both sexes but only after migration in the case of males [8].

The Sry gene, found exclusively on the Y-chromosome in normal cases, can be assessed through messenger RNA (mRNA) levels or through quantities of genomic DNA (gDNA). Although both types of assessments have been used, assessment of Sry in gDNA [13–19], which can be performed using gel electrophoresis [16, 19], is more common than assessment of Sry mRNA abundance [20, 21]. A prior report that measured Sry mRNA abundance in mouse used a PCR assay for Aard (alanine and arginine rich domain) expression to determine sex [22], but the validity of using Sry mRNA specifically to determine sex was not considered. Similarly, a prior report measured variation in Sry mRNA expression in mouse across time using RNA-sequencing, but no comparison to gDNA was made [23]. mRNA assessments benefit from evaluation of DNA that is actually transcribed, that is, by assessing the Sry mRNA that codes for the SRY protein responsible for sexual dimorphic developmental processes. These processes include the formation of a SRY and steroidogenic factor 1 (SF-1) protein complex that binds to sry-box 9 (SOX-9) enhancer regions to upregulate SOX-9 expression [24] and facilitate Sertoli cell differentiation and testis development [25]. Although some studies may also have available mRNA but not gDNA [22, 23], benefits exist for assessment of Sry from gDNA as opposed to mRNA. Unlike detection of gDNA, detection of mRNA requires the synthesis of complementary DNA (cDNA) from RNA. An additional benefit to assessing gDNA as opposed to mRNA is that gDNA is more stable because it lacks a reactive hydroxyl group found in RNA.

Despite the variety of approaches used to determine sex, few publications compare methods side-by-side. In this report, we used two methods and showed that the determination of sex using placental samples from timed-pregnant Wistar rats yielded similar results whether the approach was through assessment of gDNA or mRNA of Sry. This approach was used for the Wistar rats exposed to trichloroethylene (TCE), a common environmental contaminant, in combination with potential TCE metabolism modulators, including N-acetyl-L-cysteine (NAC) [26–28, 31] and aminooxyacetic acid (AOAA) [29, 30, 31], according to Figure 1. Samples from all experimental groups were included and appropriately analyzed to ensure treatments did not affect sex classification. Because mRNA is transcribed from DNA, we hypothesize that Sry detection from mRNA and gDNA will be congruent and successfully differentiate between male and female fetuses.

Figure 1:

Treatment schedule of the timed-pregnant Wistar rats. With gestational day (GD) 0 designated as day of copulation, rats arrived on GD 2, were trained to eat the vanilla wafer on GD 3, and were euthanized on GD 16. The following dosages of each chemical were used: 200 mg NAC/kg/day, 20 mg AOAA/kg/day, 480 mg TCE/kg/day.

Materials and Methods

Chemicals and reagents

TCE, NAC, and AOAA (purchased as O-(Carboxymethyl)hydroxylamine hemihydrochloride) were from Sigma-Aldrich (St. Louis, MO). Vanilla wafers (Nabisco) were purchased locally.

Timed-pregnant Wistar rats

Timed-pregnant Wistar rats between 60 to 90 days of age were purchased from Charles River Laboratories (Portage, MI). The day of copulation was designated as gestational day (GD) 0. The rats were transported to the University of Michigan School of Public Health animal facility on GD 2. Rats were individually housed in a controlled environment with a 12-hour light/dark cycle and provided with standard rat chow (Purina 5001) and water ad libitum.

Exposures

The placental samples used in this analysis were obtained from a toxicology study in which rats were exposed to TCE alone or in combination with chemical modulators of TCE metabolism as shown in Figure 1 using the vanilla wafer method of exposure developed by Seegal et al. [32]. The TCE dosage of 480 mg/kg/day was chosen because similar dosages induce oxidative stress and selective neurodegeneration in rats [33–35]. Importantly, one of those studies used timed-pregnant Wistar rats exposed from GD 6 to GD 16 [34]. The dosage of 480 mg/kg/day is also within an order of magnitude of the Occupational Safety and Health Administration (OSHA) Permissible Exposure Level (PEL) for inhalation exposure over an eight-hour work day [36]. The NAC dosage of 200 mg/kg/day was chosen because that is representative of effective in vivo repeated NAC exposures [37–39], including one study performed on pregnant Sprague-Dawley rats [37]. The AOAA dosage of 20 mg/kg/day is a moderate value in the range of effective AOAA dosages performed in prior in vivo rat studies [40, 41]. Assignment of rats to treatment group within each arrival (batch) was done randomly and is indicated in Supplemental Table 1. This research was approved under the Animal Welfare Assurance Number A3114–01, and the IACUC approval number PRO00006721.

Placental sample collection

Placental samples were collected for RNA and gDNA extraction on GD 16. For RNA extraction, placental samples obtained after rat euthanasia were stored in RNAlater (Qiagen, Germantown, MD) overnight at 4°C. The next day, the RNAlater was removed, and the placenta samples were stored at −80°C until RNA extraction. For gDNA extraction, the placental samples were snap-frozen in liquid nitrogen and then archived at −80 °C until gDNA extraction.

Extraction of gDNA and RNA

The gDNA was extracted from placental tissue using a NucleoSpin™ Tissue kit (Machery-Nagel, Bethlehem, PA) according to the manufacturer’s instructions. To extract RNA, placental tissue was first homogenized using a FastPrep-24 tissue and cell lyser (MP Biomedicals, Solon, OH). Homogenization occurred in RLT Buffer PLUS (Qiagen, Germantown, MD) containing 1% (v/v) 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). Homogenized placenta was then subject to RNA extraction using an RNeasy Plus Mini kit (Qiagen, Germantown, MD) according to the manufacturer’s instructions. Concentration and purity of DNA and RNA were verified using a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). DNA was stored at −20°C and RNA was stored at −80°C until further analysis.

Quantitative real-time polymerase chain reaction (qRT-PCR) of Sry

To synthesize cDNA from the RNA samples, an iScript™ cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA) was used with a Bio-Rad CFX Connect™ Real-Time System according to the manufacturer’s protocol. The protocol on the Real-Time System was as follows: (1) 25°C for 5 minutes, (2) 42°C for 30 minutes, (3) 85°C for 5 minutes, and (4) cool down to 4°C. The cDNA was stored at −20°C until further use. The gDNA extracted from placenta was diluted in elution buffer (5 mM Tris/HCl, pH 8.5) as appropriate to ensure that a given volume resulted in the same mass of gDNA added into a given qRT-PCR mixture.

qRT-PCR was performed using a total 25 µL mixture consisting of the following: 60% (v/v) SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad Laboratories, Hercules, CA), 0.32 µM of each (forward and reverse) primer, and either 40 ng of cDNA template or 50 ng of gDNA. Beta-2-microglobulin (B2m) served as the reference gene. Primer sequences and sources are listed in Supplemental Table 2. All primers were synthesized and made by Integrated DNA Technologies (Skokie, IL).

Samples were analyzed in Hard-Shell® 96-well plates (Bio-Rad Laboratories, Hercules, CA) using a Bio-Rad CFX Connect™ Real-Time System with the following protocol: (1) 95°C for 10 minutes, (2) 95°C for 15 seconds, (3) 60°C for 1 minute, (4) repeat 39 times steps 2 and 3, (5) 95°C for 1 minute, (6) 65°C for 2 minutes, (7) 65°C to gradual increase to 95°C, stopping at every 0.5°C interval for 5 seconds each. Analysis was performed using the ΔΔCt method [42], and all samples were run and analyzed in duplicate. From this procedure, we obtained Cq (also known as Ct) values [43] (and SEM), which are proportional to the number of amplification cycles necessary to achieve a threshold fluorescence and inversely proportional to the logarithm (base 2) of cDNA or gDNA template quantity [42]. We also obtained Sry (Sry/B2m) values for each sample. Although these normalized values are traditionally referred to as expression values in the case of mRNA [42], because gDNA is not expressed [44], these values will be referred as Sry (Sry/B2m) values or Sry/B2m abundance values when gDNA is included in the relevant text. Unlike the Sry Cq value, these Sry (Sry/B2m) values for both gDNA and mRNA take normalization by B2m into account.

Statistical analysis

Because each sample was run in duplicate, mean Sry and B2m Cq values and Sry expression values were obtained for each sample. Statistical analysis was performed on the mean Cq and expression values. The SEM of Cq and expression values are displayed in Supplemental Tables 3 and 4 and served to ensure accuracy of pipetting. Rat 17, Placenta 3, which had a strong Sry signal from gDNA analysis and no Sry signal from mRNA analysis, was excluded from all statistical analysis requiring sex classification. Statistical tests included student’s two-tailed unpaired t-test, two-tailed Mann-Whitney test, and one-way ANOVA followed by Tukey’s post-hoc comparison of means, as appropriate. The Mann-Whitney test was selected as a non-parametric test for data that failed to meet assumptions for parametric analysis based on its suitability for analysis of data sets with tied and zero values. Statistical tests are described in figure legends, and p<0.05 was considered statistically significant. Sry Cq values ≥ 40 automatically received a corresponding zero, or N/A, Sry/B2m value.

Results

Use of Sry (Sry/B2m) values to identify sex in placental gDNA and mRNA

The sex determined through Sry (Sry/B2m) mRNA expression matched the sex determined by Sry (Sry/B2m) value in gDNA for 90 out of 91 samples (Supplemental Table 3). There was one exception, which was positive for Sry in gDNA but negative for Sry mRNA expression (Supplemental Table 3, Rat 17, Placenta 3). Because we could not classify the latter placenta as male or female, this placenta was excluded from all subsequent statistical analyses comparing values between sexes. Among the 90 samples with concordant Sry gDNA and mRNA findings, gDNA Sry (Sry/B2m) values were 832.0% higher in males compared to females (p<0.0001) (Table 1). Similarly, mRNA Sry (Sry/B2m) expression values were all above zero for males but were all zero, resulting from Sry Cq values all ≥ 40, for females (p<0.0001) (Table 1). Furthermore, for both gDNA and mRNA analyses, the Sry (Sry/B2m) value range for female did not overlap with the Sry (Sry/B2m) value range for male (Table 1). Implicit from the lack of overlapping values, cut-offs could be assigned (Males: >0.05 and Females: <0.05 in the case of gDNA; Males: >0.01 and Females <0.01 in the case of mRNA) (Table 1), though these cannot be assumed to be universal for all studies.

Table 1:

Sex determination for rat placenta based on Sry (Sry/B2m) values for gDNA and mRNA analyses with all treatment groups combined.^a

Analysis	Sex	Mean ± SEM	Range	Cut-off	p-value
gDNA	Male	1.362 ± 0.1323	0.10017 to 6.36962	>0.05	<0.0001
gDNA	Female	1.635 × 10−3 ± 4.868 × 10−4	N/A (or 0)b to 0.01917	<0.05
mRNA	Male	1.716 ± 0.4422	0.07279 to 20.19149	>0.01	<0.0001
mRNA	Female	N/A (or 0)	N/A (or 0)b to N/A (or 0)b	<0.01

^aDescriptive statistics are shown and highlight that no values overlapped between males and females. Hence, cut-offs were determined as indicated. Two-tailed Mann-Whitley tests were used to determine p-values in the male and female comparisons. N=46 and 44 for male and female samples, respectively, for both gDNA and mRNA samples.

^bA value of N/A (or 0) is derived from the corresponding Sry Cq value of ≥ 40.

Analyses of Sry (Sry/B2m) values across and by treatment groups for gDNA and mRNA

Because the placentas were collected from rats receiving different treatments, we also analyzed the results by treatment group to ensure that treatments did not affect sex classification. Using the sex classified by both gDNA and mRNA, Table 2 contained the breakdown of sex as a function of treatment groups. Each treatment group contained at least six males and at least four females. Within treatment group, the differences in Sry (Sry/B2m) values between males and females were significant for all comparisons (p-value range of 0.0001 to 0.0095 for gDNA and p-value range of <0.0001 to 0.0095 for mRNA) (Table 3). Therefore, Sry (Sry/B2m) sex differences remained significant when analyzing by treatment group with decreased sample size, suggesting the suitability of using Sry (Sry/B2m) values for determining sex for subsets of a population, as well. Additionally, no statistically significant differences were detected in Sry (Sry/B2m) values when investigating across treatment groups for a given sex for both mRNA and gDNA (Figure 2A–C).

Table 2:

Distribution of rat placenta by treatment group and sex classification based on Sry/B2m.^a

	Number of placenta (Number of dams)
Treatment	Male	Female
Control	6 (4)	12 (4)
NAC	10 (4)	6 (4)
AOAA	6 (3)	4 (3)
TCE	9 (4)	7 (4)
TCE + NAC	8 (4)	8 (4)
TCE + AOAA	7 (4)	7 (4)
Total	46 (23)	44 (23)

^aEach dam within a treatment group came from a different batch. For 90 total samples (46 male + 44 female), sex determined via gDNA analysis matched the sex determined via mRNA analysis.

Table 3:

Sry (Sry/B2m) values within each treatment group for gDNA and mRNA analysis of rat placenta.

Analysis	Treatment	Male Sry/B2m (Mean ± SEM)	Female Sry/B2m (Mean ± SEM)	p-valuea
gDNA	Control	1.420 ± 0.1460	7.258 × 10−4 ± 2.951 × 10−4	0.0001
gDNA	NAC	1.085 ± 0.1583	3.752 × 10−3 ± 3.086 × 10−3	0.0002
gDNA	AOAA	1.886 ± 0.9149	9.275 × 10−4 ± 5.243 × 10−3	0.0095
gDNA	TCE	1.673 ± 0.1342	3.356 × 10−3 ± 1.022 × 10−3	0.0002
gDNA	TCE + NAC	1.158 ± 0.1826	1.199 × 10−3 ± 7.722 × 10−4	0.0002
gDNA	TCE + AOAA	1.090 ± 0.1489	5.600 × 10−4 ± 1.852 10−4	0.0006
mRNA	Control	1.867 ± 0.5535	0	<0.0001
mRNA	NAC	0.9734 ± 0.2341	0	0.0002
mRNA	AOAA	1.319 ± 0.4043	0	0.0095
mRNA	TCE	1.271 ± 0.3541	0	0.0002
mRNA	TCE + NAC	4.441 ± 2.304	0	0.0002
mRNA	TCE + AOAA	0.4455 ± 0.08977	0	0.0006

^ap-values were computed using the two-tailed Mann-Whitney test to compare males and females. Sample sizes are shown in Table 2.

Figure 2:

Sry/B2m values across different treatment groups separated by sex for gDNA and mRNA. Sry/B2m values for (A) male gDNA samples, (B) male mRNA samples, and (C) female gDNA samples from rat placenta. Note the different range of units of the y-axis across the different graphs and that no values between males and females overlapped. Statistically significant treatment effects were not detected with one-way ANOVA. Columns represent mean ± SEM, with individual data points shown. There is no graph for female mRNA because all female mRNA Sry/B2m values were zero. Sample sizes are shown in Table 2.

Use of Sry Cq values to identify sex for placental gDNA and mRNA

To provide insight for studies not using a traditional reference gene or using a reference gene other than B2m, we evaluated non-normalized Sry Cq mRNA and gDNA values as a metric for distinguishing between male and female samples, as well. As a method for classification of sex, this would require that the nucleic acids underwent accurate measurement and pipetting to assure equal amounts of genetic material in the sample wells.

Evaluation and analysis of Sry Cq values were performed on the basis of sex classification determined by the cut-offs described in Table 1. For mRNA, all classified female samples had Sry Cq values that were not determined (ND) (listed as “N/A” in the Bio-Rad CFX program and applied to sample readings ≥ 40) whereas all males had a defined Sry Cq value (Table 4 and Supplemental Table 4). Although statistical analysis could not be performed because Sry Cq values were not determined for all females, there was an obvious difference between male and female Sry Cq values. In contrast, all males had an existent Sry Cq gDNA value and most females also had an existent Sry Cq gDNA value (Table 4 and Supplemental Table 4). A statistically significant difference was observed between males and the subset of females with Sry Cq gDNA values (excluding four females with undetermined Cq values) (p<0.0001). However, there was an overlap in range between male and female Sry Cq values for this gDNA analysis (the highest Sry Cq value for males was 31.08; the lowest Sry Cq value for females was 30.27, and three females had a Sry Cq value below 31.08) (Table 4). Thus, whereas Sry Cq values were observed to be different between males and females for both mRNA and gDNA (Table 4), Sry Cq values without normalization to a reference gene could lead to false sex classification. Overall, Sry Cq values could benefit in accuracy from normalization or assessment of a secondary metric, although the reliability of Sry Cq value assessment was especially true in our mRNA analysis where all females had undetermined Sry Cq values.

Table 4:

Rat placental Sry Cq gDNA and mRNA non-normalized values with all treatment groups combined.^a

Analysis	Sex	Sry Cq (Mean ± SEM)	Sry Cq Range	p-valueb
gDNA	Male	24.56 ± 0.3201	21.92 to 31.08	<0.0001
gDNA	Female	34.52 ± 0.3609c	30.27 to N/Aa
mRNA	Male	33.92 ± 0.2431	30.32 to 37.17	N/Aa
mRNA	Female	N/Aa	N/Aa

^aThe Bio-Rad CFX program denoted Cq values ≥ 40 as N/A.

^bThe gDNA data were analyzed using a two-tailed unpaired t-test to compare male and female Cq values (excluding four female samples with undetermined Sry Cq values). For mRNA samples, there was no overlap between male and female Cq values, but statistical analysis was not performed because all female samples had undetermined Sry Cq values (≥ 40). Sample sizes are indicated in Table 2.

^cThis particular mean ± SEM calculation excluded the four female samples with undetermined Sry Cq values.

Analyses of Sry Cq values across and by treatment group for gDNA and mRNA

Analyses of Sry Cq within and across treatment groups were conducted to determine the suitability of Sry Cq values for sex determination specific to treatment groups. Because all females had undetermined Sry Cq mRNA values and all males had defined Sry Cq mRNA values, this was true for any given treatment group, also (Table 5), and the Sry Cq mRNA values classified males and females for all treatment groups consistent with Sry (Sry/B2m) analysis. For gDNA samples, differences between sex were statistically significant for all treatment groups (p-value range from <0.0001 to 0.0066; Table 5). Furthermore, the mRNA-derived Sry Cq values for male samples did not statistically differ across treatment groups (Figure 3B). However, several significant differences were observed between treatment groups for non-normalized male and female Sry Cq gDNA values (Figures 3A and 3C, respectively), in contrast to the lack of treatment-related differences when treatment groups were combined (Table 4). Nonetheless, sex differences within each of the treatment groups (Table 5) were still observed. This further indicates that investigation of Sry data normalized to B2m (i.e., using a reference gene), in which no statistical differences were observed (Figure 2A–C), may be more suitable for avoiding treatment-related discrepancies in sex classification.

Table 5:

Rat placental Sry Cq values within each treatment group for non-normalized gDNA and mRNA.^a

Analysis	Treatment	Male Sry Cq Value (Mean ± SEM)	Female Sry Cq Value (Mean ± SEM)	p-valueb
gDNA	Control	23.18 ± 0.2562	34.89 ± 0.5491	<0.0001
gDNA	NAC	24.70 ± 0.4034	34.55 ± 1.031	<0.0001
gDNA	AOAA	26.98 ± 1.136	34.70 ± 1.768c	0.0066
gDNA	TCE	22.51 ± 0.1446	32.02 ± 0.4172	<0.0001
gDNA	TCE + NAC	25.53 ± 0.9616	35.32 ± 1.011d	<0.0001
gDNA	TCE + AOAA	25.03 ± 0.488	35.76 ± 0.735c	<0.0001
mRNA	Control	34.05 ± 1.013	N/Aa	N/Aa
mRNA	NAC	33.97 ± 0.5436	N/Aa	N/Aa
mRNA	AOAA	33.36 ± 0.5146	N/Aa	N/Aa
mRNA	TCE	34.00 ± 0.3888	N/Aa	N/Aa
mRNA	TCE + NAC	33.76 ± 0.5704	N/Aa	N/Aa
mRNA	TCE + AOAA	34.28 ± 0.7675	N/Aa	N/Aa

^aThe Bio-Rad CFX program denoted Cq values ≥ 40 as N/A.

^bp-values comparing males to females were computed using unpaired two-tailed t-tests. Sample sizes are shown in Table 2.

^cThese particular mean ± SEM calculations excluded one female sample with undetermined Sry Cq values.

^dThis particular mean ± SEM calculation excluded two female samples with undetermined Sry Cq values.

Figure 3:

Sry Cq values across different treatment groups separated by sex for gDNA and mRNA. Sry Cq values for (A) male gDNA samples, (B) male mRNA samples, and (C) female gDNA samples from rat placenta. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc multiple comparison of means. Non-overlapping letters signify statistical significance. Columns represent mean ± SEM, with individual data points shown. There is no graph for female mRNA because all female Sry Cq values derived from mRNA analysis were undetermined (≥ 40). Sample sizes are shown in Table 2.

The validity of B2m as a reference gene

B2m was used as the reference gene in this project. Presence of B2m ensured that nucleic acid was present in the samples and that lack of Sry Cq or expression was not due to degraded nucleic acid but rather because the sample was female. We obtained strong Cq values for B2m for all samples, particularly in the case of mRNA where the Cq for B2m averaged 19.94 (the Cq for B2m in the case of gDNA analysis averaged 25.37). No significant differences between sexes were observed for B2m Cq gDNA or mRNA values whether combined or separated by treatment group (Tables 6 and 7, respectively). Supplemental Table 4 contains the results by individual sample.

Table 6:

Rat placental B2m Cq values for gDNA and mRNA with all treatment groups combined.

Analysis	Sex	Sry Cq (Mean ± SEM)	Sry Cq Range	p-valuea
gDNA	Male	25.58 ± 0.2372	23.44 to 30.44	0.1879
gDNA	Female	25.16 ± 0.2108	22.71 to 28.34
gDNA	Male and Female	25.37 ± 0.1598	22.71 to 30.44
mRNA	Male	19.79 ± 0.1734	17.87 to 23.42	0.2417
mRNA	Female	20.11 ± 0.2103	18.13 to 24.19
mRNA	Male and Female	19.94 ± 0.136	17.87 to 24.19

^ap-values were computed using the two-tailed Mann-Whitney test to compare males and females. Sample sizes are shown in Table 2.

Table 7:

Rat placental B2m Cq values within each treatment group by sex for gDNA and mRNA.

Analysis	Treatment	Male B2m Cq Value (Mean ± SEM)	Female B2m Cq Value (Mean ± SEM)	p-valuea
gDNA	Control	24.51 ± 0.2894	24.08 ± 0.2391	0.2996
gDNA	NAC	25.67 ± 0.3888	25.34 ± 0.4829	0.6062
gDNA	AOAA	27.32 ± 0.8443	26.31 ± 1.009	0.4663
gDNA	TCE	24.03 ± 0.1258	24.11 ± 0.2303	0.7481
gDNA	TCE + NAC	26.18 ± 0.6365	26.11 ± 0.4115	0.9316
gDNA	TCE + AOAA	26.17 ± 0.2715	26.13 ± 0.3380	0.9178
mRNA	Control	20.94 ± 0.4439	20.36 ± 0.3831	0.3738
mRNA	NAC	19.31 ± 0.2810	19.73 ± 0.2664	0.3310
mRNA	AOAA	19.56 ± 0.1875	19.37 ± 0.2849	0.5635
mRNA	TCE	19.30 ± 0.2362	19.56 ± 0.1880	0.4332
mRNA	TCE + NAC	20.55 ± 0.6467	20.53 ± 0.7690	0.9893
mRNA	TCE + AOAA	19.43 ± 0.2447	20.47 ± 0.6602	0.1638

^ap-values were computed using the two-tailed Mann-Whitney test to compare males and females. Sample sizes are shown in Table 2.

We also investigated B2m Cq values across treatment groups, separated by sex (Figure 4). Whereas multiple comparisons within gDNA analysis were statistically significant (Figures 4A and 4C), only one comparison within mRNA analysis was statistically significant (Figures 4B and 4D). Although these differences exist, they should not be overemphasized. Despite these differences among B2m Cq values across treatment groups (particularly for gDNA more than mRNA), the Sry (Sry/B2m) values yielded classification of male versus female for both gDNA and mRNA analyses in which both analyses agreed with each other for 90 out of 91 samples. Although B2m Cq differences could have contributed variability to the Sry (Sry/B2m) values, because classification of sex based on Sry (Sry/B2m) values led to gDNA and mRNA agreement for 90 out of 91 samples with clear cut-offs (Table 1), inclusion of B2m as a reference gene increased validity of sex classification based on Sry.

Figure 4:

B2m Cq values across different treatment groups separated by sex for gDNA and mRNA. B2m Cq values for (A) male gDNA samples, (B) male mRNA samples, (C) female gDNA samples, and (D) female mRNA samples from rat placenta. Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc multiple comparison of means. Statistical significance is denoted by non-overlapping letters. Columns represent mean ± SEM, with individual data points shown. Sample sizes are shown in Table 2.

Discussion

We identified qRT-PCR as a method for fetal sex determination producing remarkably similar results whether analyzing gDNA or mRNA for Sry. A strength of our study comes from assessment of Sry (Sry/B2m) values, Sry Cq values, and validity of our reference gene, B2m, for both gDNA and mRNA samples. This allowed inferences about the suitability of each metric, particularly Sry (Sry/B2m) values or Sry Cq values for sex determination, aspects of which have been used in sex determination from gDNA in prior reports.

The most useful metric used in the present study to determine sex was the Sry (Sry/B2m) metric. Assessment of this metric allowed sex classification in which the classification via gDNA matched the classification for mRNA for 90 out of 91 of the samples. For both mRNA and gDNA, no Sry (Sry/B2m) values classified as male overlapped those classified as female. We were also able to establish cut-offs of Sry (Sry/B2m) values by sex. Finally, the Sry (Sry/B2m) metric also benefits from assurance of being normalized to a reference gene.

The use of only Sry Cq values to evaluate sex has advantages and limitations. Xiang et al. (2016) evaluated the ratio of AR to Sry to determine sex in Wistar rats and observed that the Sry Cq values were much lower in males compared to females [19]. In our mRNA analysis, all our females had non-defined Sry Cq values (anything ≥ 40) whereas males had Sry Cq values ranging from 30.32 to 37.17 (Table 3), clearly distinguishing the difference between male and female placentas. The difference in gDNA Sry Cq values between sexes was evident despite three females having a lower Sry Cq than the male with the highest Sry Cq value. Overall, Sry Cq mRNA values could be used to determine sex because these values were always undetermined for the female placentas.

Although B2M/B2m is a relatively common reference gene used for placental mRNA analysis [45–47], some considerations arose from our study. First, the most rigorous qRT-PCR experiments would benefit from use of three or more reference genes [48]. However, the quantity of samples we used and the analysis of both gDNA and mRNA limited our ability to include additional reference genes. Additionally, because we were not primarily interested in the degree of Sry expression, the use of additional reference genes for this study was not as relevant as for an experiment assessing degree of gene expression. Secondly, although a few differences in B2m Cq values were detected between treatment groups, particularly for gDNA, our B2m Cq values were strong. Furthermore, our use of Sry (Sry/B2m) values, which were normalized to B2m, resulted in sex classification in which mRNA and gDNA analysis agreed for 90 out of 91 samples.

A strength of our study is the analysis of samples with treatment groups combined as well as separated by treatment group. This allowed us to determine that the Sry (Sry/B2m) and Sry Cq value differences were upheld for the totality of samples and by treatment group. Therefore, not only do we recommend the use of Sry (particularly (Sry/B2m)) values from either gDNA or mRNA analyses in determining fetal sex, but we also suggest that Sry values from either gDNA or mRNA analyses are relevant to both a population with varied treatments and a more homogenous population. Importantly, this is of interest to researchers determining placental sex in a population that is totally unexposed or another more homogenous population compared to the totality of the samples we used in our analysis. A worthy future direction could be to see the applicability of this in different strains of rats (i.e., Wistar rats versus Sprague-Dawley rats) or different species (i.e., mice).

One of our placentas had a strong Sry signal from gDNA analysis but lacked a Sry signal from mRNA analysis. The possibility exists that the gDNA corresponding to Sry had not been transcribed to mRNA at the time of rat euthanasia, GD 16. Alternatively, the placenta may have lacked crucial components of the machinery required to transcribe Sry gDNA into mRNA. A less likely explanation is that only a portion of the placenta contained Sry gDNA or mRNA; that is, the distribution of Sry across given placenta may have been uneven. However, we analyzed separately various portions of the stored placenta for gDNA and mRNA and arrived at the same classification each time. Additionally, because this placenta was the only placenta out of 91 placentas in which sex was not definitively assigned because of differing gDNA and mRNA analysis classifications, the aforementioned phenomenon is unlikely to be widespread in Wistar rat Sry gDNA or mRNA.

The current study is not without limitations. The first limitation is that the current study investigated placental gDNA and mRNA at only one time point, GD 16. In mice, Sry mRNA varies in abundance as a function of gestational age [23]. Particularly, in a pool of mice gonadal tissue, Sry mRNA expression for male samples are at a low level on 10.5 days post coitum (DPC), peak on 11.5 DPC, and decreases back to a low level on 12.5 DPC [23]. The second limitation is that the current study used only two genes, Sry and B2m. To improve rigor and reproducibility, further studies could include additional genes with relevance to both males and females, such as Kdm5c and Kdm5d [49], and additional gestational ages.

In summary, we established that gDNA or mRNA from placenta of Wistar rats at GD 16 allowed similar sex classification when assessing Sry (Sry/B2m) values. This was true whether the samples were separated by treatment group or not. Assessment of Sry Cq values by themselves indicated that the male values were significantly different from the female values regardless of separation by treatment group. However, because of some overlap between male and female Sry Cq values, limitations and alternative approaches should be kept in mind if using only the Sry Cq metric for sex determination. Assessment of B2m Cq values indicated that our reference gene provided strong and low Cq values, particularly for mRNA. As a whole, we have established that sex classification via Sry mRNA, which has not been assessed in conjunction with Sry gDNA previously, was a valid method of sex classification compared with Sry gDNA, the method used more extensively in sex determination in prior studies.