International Journal of Molecular Sciences Article Testis-Specific Protein Y-Encoded (TSPY) Is Required for Male Early Embryo Development in Bos taurus Na-Young Rho 1 , Teresa Mogas 2 , W. Allan King 1,3 and Laura A. Favetta 1, * 1 Reproductive Health and Biotechnology Lab, Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada 2 Department of Medicine and Animal Surgery, Autonomous University of Barcelona, Cerdanyola del Vallés, 08193 Barcelona, Spain 3 Karyotekk Inc., Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada * Correspondence: lfavetta@uoguelph.ca Abstract: TSPY is a highly conserved multi-copy gene with copy number variation (CNV) among species, populations, individuals and within families. TSPY has been shown to be involved in male development and fertility. However, information on TSPY in embryonic preimplantation stages is lacking. This study aims to determine whether TSPY CNV plays a role in male early development. Using sex-sorted semen from three different bulls, male embryo groups referred to as 1Y, 2Y and 3Y, were produced by in vitro fertilization (IVF). Developmental competency was assessed by cleavage and blastocyst rates. Embryos at different developmental stages were analyzed for TSPY CN, mRNA and protein levels. Furthermore, TSPY RNA knockdown was performed and embryos were assessed as per above. Development competency was only significantly different at the blastocyst stage, with 3Y being the highest. TSPY CNV and transcripts were detected in the range of 20–75 CN for 1Y, 20–65 CN for 2Y and 20–150 CN for 3Y, with corresponding averages of 30.2 ± 2.5, 33.0 ± 2.4 and 82.3 ± 3.6 copies, respectively. TSPY transcripts exhibited an inverse logarithmic pattern, with 3Y showing significantly higher TSPY. TSPY proteins, detected only in blastocysts, were not significantly different among groups. TSPY knockdown resulted in a significant TSPY depletion (p < 0.05), with Citation: Rho, N.-Y.; Mogas, T.; King, no development observed after the eight-cell stage in male embryos, suggesting that TSPY is required W.A.; Favetta, L.A. Testis-Specific for male embryo development. Protein Y-Encoded (TSPY) Is Required for Male Early Embryo Development in Bos taurus. Int. J. Mol. Keywords: copy number variation (CNV); sex-sorted semen; in vitro fertilization; male development; Sci. 2023, 24, 3349. https://doi.org/ testis-specific protein Y-encoded (TSPY) 10.3390/ijms24043349 Academic Editors: Suresh C. Sikka, Saradha Baskaran, Manesh 1. Introduction Kumar Panner Selvam and Omer A. Raheem In humans, 20–30% of infertility is male-related, and almost 50% of these causes are idiopathic [1]. Chromosome abnormalities, microdeletion of the azoospermia factor (AZF) Received: 8 January 2023 regions of the Y chromosome, genetic polymorphism and association with specific sequence Revised: 27 January 2023 repeats have been shown to affect spermatogenesis and male fertility [2–4]. Accepted: 5 February 2023 Copy number variation (CNV), structural variation of the genomic sequence by dele- Published: 8 February 2023 tion, insertion and duplication resulting in different number of copies of a gene, is associated with genetic diversity among individuals and species [5,6]. Studies have shown that an increased or decreased number of gene copies results in abnormal recombination and Copyright: © 2023 by the authors. meiosis, leading to germ cell loss which further results in infertility [7]. CNV in the male Licensee MDPI, Basel, Switzerland. specific region of the Y chromosome (MSY) is a result of rearrangement and duplication, This article is an open access article which are known to occur frequently as a consequence of intrachromosomal interactions distributed under the terms and of non-allelic homologous recombination (NAHR) that alter the amplification, deletion or conditions of the Creative Commons gene conversion [8,9]. Attribution (CC BY) license (https:// Testis-specific protein Y-encoded (TSPY) is a male specific gene that is located in the creativecommons.org/licenses/by/ MSY [10,11], mostly on the ampliconic region of the Yp (DYZ5) near the centromere, but 4.0/). with evidence of also being on the Yq arm [12–14]. TSPY is a highly conserved multi-copy Int. J. Mol. Sci. 2023, 24, 3349. https://doi.org/10.3390/ijms24043349 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 3349 2 of 18 gene with CNV among species, populations, individuals and within families. Rats have only one functional TSPY copy [15], while mice have a single pseudogene [16]. Pigs have three copies but are not CN variant [17]. Humans have 30–60 [18] TSPY CN, and the largest range of variation can be observed in cattle with a range of 20–200 copies [19]. TSPY CN is associated with male specific functions. In humans, higher TSPY CN was present in infertile men and men with prostate cancer [18]. In bovines, bulls with higher TSPY CN showed higher fertility rates [20], whereas low TSPY CN was linked to bulls with inferior semen production [21]. TSPY CN shows temporal changes during aging both in vitro and in vivo [22], suggesting possible epigenetic recombination of TSPY. TSPY is expressed exclusively in the germ cells during embryogenesis and spermatogenesis and is present in both prenatal and adult testis [13,23]. TSPY shows transcript heterogeneity which codes over 290 amino acids and allows alternative splicing patterns that result in a variety of polymorphic proteins [11,13,24]. Importantly, TSPY protein is conserved within the SET/NAP domain that interacts with the Cyclin B-binding domain [13] and plays an important role in promoting differentiation and proliferation during spermatogenesis, especially during spermatonoia proliferation, self-renewal, and at spermatocyte differen- tiation at prophase I [25,26]. TSPY levels are also associated with gonadoblastoma, and the tumorigenic capability in gonad and prostate cancers [27–29]. Most TSPY studies have been conducted in adult and fetal tissues and cells, but not much is known about the earlier preimplantation stages of development, which are key for fertility [30], although TSPY expression has been shown at the bovine blastocyst stage. There is substantial evidence that copy number varies among brothers and changes with aging [22]. However, in early embryos, it has only been superficially examined [30,31]. Therefore, the aim of the present study is to investigate whether TSPY is present and expressed and to determine its function during bovine preimplantation male embryo development for use as a translational model for humans [32]. 2. Results 2.1. Early Embryo Developmental Percentage Cleavage and blastocyst rates from groups produced by sex-sorted semen from the three different bulls and the unsorted semen (control) from the conventional bull semen are shown in Table 1 and Figure 1A,B. No significant differences in cleavage rate between oocytes fertilized with control semen and Y-bearing sperm were detected (Figure 1A). However, the cleavage rate of oocytes fertilized with X-chromosome bearing sperm was significantly lower than the control and Y-chromosome bearing groups (p < 0.05). There were no significant differences among the female embryo groups nor among the male embryo groups. Specifically, the average cleavage rates in the combined X-bearing sperm group and the combined Y-bearing sperm group were 62.2 ± 3.8 and 78.6 ± 3.5, respectively. The overall blastocyst rate among the embryos derived from the Y-bearing sperm was almost double that of the X-bearing sperm group (29.0 ± 1.7 vs. 16.6 ± 2.0) (Figure 1B). The X- bearing groups were not significantly different from each other, but all were significantly lower than the control group and the Y-bearing groups. Two groups derived from the Y- bearing sperm were significantly (p < 0.05) lower than the control group and the 3Y-bearing group. Blastocysts from all groups were examined for morphology and total blastocyst cell number, which were normal and within the range of 110–130 blastomeres/blastocysts, respectively, with no significant differences among the groups. Int. J. Mol. Int. Sci. J. Mol. 2023, Sci. 24,24, 2023, 3349 x FOR PEER REVIEW 33ofof1818 Table 1. Developmental percentage and total cell number from IVP embryos produced from unsorted Table 1. Developmental percentage and total cell number from IVP embryos produced from un- and sex-sorted sorted bull semen. and sex-sorted bull semen. Oocytes Oocytes Cleavage Cleavage Blastocysts Blastocysts Blastocyst Blastocyst Cell Cell Number Number Groups Groups No.No. No. No. Percentage Percentage (%) (%) No.No. Percentage Percentage (%) (%) No. No. Mean±±SEM Mean SEM 1X1X 356 356 210 210 58.9 3.6 ±a 3.6 36 36 17.1 ± ±2.22.2 15 115.4±± 15.1 15.1 a a a 58.9 ± 17.1 15 115.4 2X 2X 298 298 182 182 61.0 61.0 ± 4.7 ±a 4.7 a 30 30 16.5 ± 1.7 a 16.5 ± 1.7 a 15 15 121.1 ± 12.4 121.1 ± 12.4 3X 314 210 66.9 ± 3.2 a 34 16.2 ± 2.0 a 15 124.7 ± 9.3 3X 314 210 66.9 ± 3.2 a 34 16.2 ± 2.0 a 15 124.7 ± 9.3 1Y 416 316 75.9 ± 3.4 b 84 26.6 ± 1.6 b 15 120.7 ± 14.0 1Y 416 316 75.9 ± 3.4 b 84 26.6 ± 1.6 b b 15 120.7 ± 14.0 2Y 460 362 78.6 ± 3.4 b 90 24.9 ± 1.9 15 116.1 ± 10.5 2Y3Y 460 534 362 430 78.6 ± 3.4 ±b 3.5 b 80.5 90 146 24.9 ±± 34.0 b c 1.91.6 15 15 119.6±±10.5 116.1 7.4 c c Con 3Y 534 179 430 144 80.5 ± 3.5 ±b 3.2 b 80.4 146 50 ± ±1.61.6 34.7 34.0 15 15 122.3±± 7.4 119.6 8.6 Con 179 144 Values in80.4 the±same 3.2 b column with 50 different superscript 34.7 ± 1.6 letters c indicate 15 significant differences 122.3 ± 8.6 (p < 0.05). Values in the same column with different superscript letters indicate significant differences (p < 0.05). A B Cleavage rate Blastocyst rate 100 40 c c b b b b 80 Blatocyst rate (%) Cleavage rate (%) a 30 b b a a 60 a a a 20 40 10 20 0 0 1X 2X 3X 1Y 2Y 3Y Cont 1X 2X 3X 1Y 2Y 3Y Cont Groups Groups Figure 1. Developmental percentage of early embryos produced from unsorted and sex-sorted (X- and Y-bearing) Figure semen from bulls 1. Developmental 1, 2 and percentage of 3.early (A) represents the percentage embryos produced of cleavage from unsorted in sex-sorted and each female(X- and (1X, Y-bearing) 2X, 3X), malesemen from (1Y, 2Y, 3Y)bulls 1, 2 andgroups. and control 3. (A) represents the percentage (B) represents of cleavage the percentage in each female of the blastocyst in (1X,female each 2X, 3X), (1X,male 2X, (1Y, 3X), 2Y, male3Y) and (1Y, 2Y,control 3Y) andgroups. control(B) represents groups. the percentage Each group of the10blastocyst were includes biologicalin each female replicates (1X, 2X, in which 3X), of a total male (1Y, 2557 2Y, 3Y) oocyte wereandused. control Thegroups. data isEach showngroup were ± as mean includes 10 biolog- SEM, different ical replicates in which a total of 2557 oocyte were used. The data is shown as mean ± superscript letters above the columns indicate significant differences within each group (p < 0.05). SEM, different superscript letters above the columns indicate significant differences within each group (p < 0.05). 2.2. TSPY Copy Number Variation (CNV) 2.2.TSPY TSPYCN Copy wasNumber Variation measured in 30 (CNV) individually collected blastocysts from male and female groupsTSPY CN3X, (1X, 2X, was1Y,measured in 30was 2Y, 3Y). TSPY individually not detected collected blastocysts in the female groupfrom (1X, male 2X, 3X)and butfe- male was groups present in(1X, 2X,male all the 3X, 1Y, 2Y, 3Y). groups (1Y,TSPY waswith 2Y, 3Y), not CNV detected in thethe between female group brother (1X, 2X, embryos 3X) but shown in was present Figure 2A–C.inTSPY all the maleranges CNV groups (1Y,20–75, were 2Y, 3Y), withand 20–65 CNV between 20–150 the2Y for 1Y, brother and 3Yembryos groups,shown in Figure respectively, 2A–C. with TSPYnumbers average CNV ranges of 30.2 ± 2.5, were 20–75, ± 2.4and 20–65 33.0 and 82.3 ± 20–150 for3.6, 1Y, respectively. The average 2Y and 3Y groups, number with respectively, TSPY CN of theaverage from the numbers of male 30.2 ±group was± significantly 2.5, 33.0 2.4 and 82.3 ± higher than the number 3.6, respectively. in the 3Y The average group (p number of