REPRODUCTIONRESEARCH

Expression of steroidogenic during equine testicular development

J Almeida, A J Conley, L Mathewson and B A Ball Department of Population Health and Reproduction, School of Veterinary Medicine, University of California Davis, Davis, California 95616, USA Correspondence should be addressed to B A Ball; Email: [email protected]

Abstract

In the mammalian testis, Leydig cells are primarily responsible for steroidogenesis. In adult stallions, the major endocrine products of Leydig cells include testosterone and . 3b-hydroxysteroid dehydrogenase/D5-D4-isomerase (3bHSD) and 17a-hydroxyl- ase/17,20-lyase (P450c17) are two key steroidogenic enzymes that regulate testosterone synthesis. produced by P450c17 serve as substrate for synthesis. The aim of this study was to investigate localization of the steroidogenic enzymes P450c17, 3bHSD, and P450arom and to determine changes in expression during development in the prepubertal, postpubertal, and adult equine testis based upon immunohistochemistry (IHC) and real-time quantitative PCR. Based on IHC, 3bHSD immunolabeling was observed within seminiferous tubules of prepubertal testes and decreased after puberty. On the other hand, immunolabeling of 3bHSD was very weak or absent in immature Leydig cells of prepubertal testes and increased after puberty. HSD3B1 (3bHSD gene) mRNA expression was higher in adult testes compared with prepubertal (PZ0.0001) and postpubertal testes (PZ0.0041). P450c17 immunolabeling was observed in small clusters of immature Leydig cells in prepubertal testes and increased after puberty. CYP17 (P450c17 gene) mRNA expression was higher in adult testes compared with prepubertal (PZ0.030) and postpubertal testes (PZ0.0318). A weak P450arom immunolabel was observed in immature Leydig cells of prepubertal testes and increased after puberty. Similarly, CYP19 (P450arom gene) mRNA expression was higher in adult testes compared with prepubertal (PZ0.0001) and postpubertal (PZ0.0001) testes. In conclusion, Leydig cells are the primary cell type responsible for and estrogen production in the equine testis. Reproduction (2011) 141 841–848

Introduction pattern of expression of HSD3B1 (3bHSD gene) and CYP17 vary between different species and throughout In the male, testosterone is required for maturation development. The study of 3bHSD and P450c17 of germ cells, maturation of sperm, and thus fertility. distribution in the testis is essential for understanding b D5 D4 3 -Hydroxysteroid dehydrogenase/ - -isomerase of testicular dynamics of androgen production. b a (3 HSD) and 17 -hydroxylase/17,20-lyase cytochrome Androgens produced by P450c17 serve as substrates P450 (P450c17) are two key steroidogenic enzymes that for estrogen synthesis. Estrogens have been shown to be regulate testosterone synthesis. 3bHSD immunolabel important in regulating steroidogenesis and spermatogen- has been observed in the smooth endoplasmic reticulum esis (Abney 1999, O’Donnell et al. 2001). (ER) of precursor Leydig cells of postnatal rat testis (P450arom) is the responsible for (Majdic et al. 1995, Haider & Servos 1998) and in both estrogen production, and its expression has been reported in Leydig and Sertoli cells of cynomolgus monkey testis the ER. The distribution of P450arom varies within testes of (Liang et al. 1998). CYP17 expression has been reported different species. P450arom has been observed in Leydig in the Leydig cells of newborn pigs (Conley et al. 1994, cells of humans (Inkster et al. 1995), boars (Conley et al. 1996) and mature boars (Sasano et al. 1989). In addition, 1996), rams (Bilinska et al. 1997), and stallions (Almadhidi Cyp17 (P450c17 gene) expression has also been 1995). In contrast, expression of P450arom in the testes of reported in different tissues including the liver (Vianello mice (Nitta et al.1993), brown bears (Tsubota et al. 1993), et al. 1997) and the stomach (Le Goascogne et al. 1995) and roosters (Kwon et al.1995) was present not only in of the rat, and its pattern of expression appears to change Leydig cells but also in spermatids inside the seminiferous with development in the mouse, in which Cyp17 is tubules. In addition, P450arom was observed in the Sertoli expressed in fetal adrenal gland and disappears in the cells of immature rat testes as well as in Leydig, Sertoli, and mature adrenal gland (Keeney et al. 1995). Therefore, the germ cells in adult rat testes (Papadopoulos et al. 1986,

q 2011 Society for Reproduction and Fertility DOI: 10.1530/REP-10-0499 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 09/26/2021 10:20:34PM via free access 842 J Almeida and others

Levallet et al. 1998). Therefore, P450arom may be present in Results at least three different cells in the testis: Sertoli cells, Leydig 3bHSD cells, and germ cells. The study of P450arom distribution is essential for the understanding of possible sites of estrogen 3bHSD immunolabeling was observed in presumptive production in the testis. Sertoli cells but not germ cells within seminiferous Even though various studies report the presence of tubules of prepubertal testes; however, 3bHSD immuno- 3bHSD (Hejmej & Bilinska 2008) and P450arom labeling was not observed in Sertoli cells after puberty (Eisenhauer et al. 1994, Almadhidi et al. 1995, Hess & (Fig. 1). Conversely, immunolabeling of 3bHSD was Roser 2004, Hejmej & Bilinska 2008) in the equine testis, very weak or absent in immature Leydig cells of there is relatively limited information concerning prepubertal testis and increased in the postpubertal changes in the expression of these steroidogenic enzymes and adult testis. In postpubertal testis, Leydig cells during testis development. Therefore, the character- stained in tight, well-defined clusters, whereas in adult ization of steroidogenic enzyme expression in the testis immuno-positive Leydig cells were scattered developing equine testis is important in understanding throughout the interstitial space. When the primary testicular steroidogenesis and cellular differentiation. antisera were omitted, no immunolabeling was The aim of this study was to investigate localization observed (Fig. 1). Consistent with the immuno- of the steroidogenic enzymes P450c17, 3bHSD, and histochemical results, HSD3B1 mRNA expression was P450arom and to determine changes in the expression higher in adult testes compared with prepubertal and during development in the prepubertal, postpubertal, postpubertal testes (Table 1). In addition, HSD3B1 and adult equine testis based upon immunohistochem- mRNA expression was higher in postpubertal than istry (IHC) and real-time quantitative PCR (RT-qPCR). prepubertal testes (Table 1).

A B

CD

Figure 1 3bHSD immunolabeling in prepubertal EF (A), postpubertal (C), and adult (E) testis with respective negative controls (B, D, and F). 3bHSD immunolabeling was observed in pre- sumptive Sertoli cells (arrows) but not in germ cells (arrowheads) inside seminiferous tubule of prepubertal testes and disappeared in the postpubertal testis. Conversely, immunolabeling of 3bHSD was very weak or absent in immature Leydig cells (bold arrows) of prepubertal testis but significantly increased by age. 50.0 µm Immunolabel was not observed when primary antisera were omitted.

Reproduction (2011) 141 841–848 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/26/2021 10:20:34PM via free access Expression of steroidogenic enzymes 843

Table 1 Expression of mRNA for HSD3B1, CYP17, and CYP19 during equine testis development based upon quantitative real-time PCR.

b DDCt Compared to Fold difference a mRNA Group group in expression S.E.M. lower CL upper CL P value HSD3B1 Prepubertal Adult 2.362207 0.3721907 1.576953 3.147461 !0.0001* Postpubertal Adult 1.422536 0.4297688 0.515803 2.329269 0.0041* Prepubertal Postpubertal 0.939671 0.3721907 0.154417 1.724925 0.0218* CYP17 Prepubertal Adult 5.062008 1.450874 1.98629 8.137723 0.0030* Postpubertal Adult 3.869864 1.645136 0.38233 7.357397 0.0318* Prepubertal Postpubertal 1.192144 1.450874 K1.88357 4.267859 0.4233 CYP19 Prepubertal Adult 8.561966 0.926344 6.60755 10.51638 !0.0001* Prepubertal Postpubertal 5.811809 0.926344 3.857393 7.76622 !0.0001* Postpubertal Adult 2.750157 1.06965 0.493392 5.00692 0.0198* a DDCt G b Fold differences in expression between groups were determined as 2 and are expressed as mean S.E.M. Cycle threshold (Ct) was determined for each target gene and the reference gene (Equine b2-microglobulin) and the DCt determined for each target gene. Confidence intervals (95%) between groups were determined by preplanned comparisons using a t-test to determine the DDCt.

P450c17 a NADH (NAD)-dependent membrane-bound enzyme that is located in the ER and mitochondria of cells P450c17 immunolabeling was observed in small clusters b of immature Leydig cells in prepubertal testes, and (Wattenberg 1958). It catalyzes the sequential 3 HSD D5 D4 D5 Leydig cell labeling for P450c17 increased markedly and - -isomerization of the - precursors , 17-hydroxypregnenolone (17-OH- in postpubertal and adult testes (Fig. 2). In postpubertal 4 testis, Leydig cells stained in tight, well-defined clusters, PREG), and DHEA into their respective D -ketosteroids: whereas in adult testis Leydig cells were stained progesterone (PROG), 17a-hydroxyprogesterone scattered throughout, in a pattern similar to that seen (17-OH-PROG), and androstenedione (ADION), for 3bHSD. A faint P450c17 immunolabel was also which are the precursors for additional enzymatic observed in the seminiferous tubule of prepubertal steps culminating in testosterone and estrogen pro- testes, which appeared to decrease in postpubertal and duction. 3bHSD has been observed in the Leydig cells adult testes. When primary antisera were omitted, no of rat testis (Majdic et al. 1995, Haider & Servos 1998) staining was observed (Fig. 2). CYP17 mRNA expression and in both Leydig and Sertoli cells of monkey testis was higher in adult testes compared with prepubertal (Liang et al. 1998). According to Liang et al. (1999), and postpubertal testes (Table 1). However, there was no 3bHSD immunolabel was detected in some Leydig significant difference between CYP17 mRNA expression cells and in Sertoli cells of neonatal, late infantile, in prepubertal and postpubertal testes (Table 1). pubertal, and adult monkey testes. In contrast, only a few Leydig cells, but no Sertoli cells, expressed immunolabel in early infantile testes. In the stallion, P450arom 3bHSD has been found in Leydig cells (Hejmej & A weak P450arom immunolabel was observed in Bilinska 2008) of adult testis. However, the pattern of immature Leydig cells of the prepubertal testis, and expression of 3bHSD during development is not well P450arom immunolabeling increased markedly in known in the horse. In this study, 3bHSD immunolabel Leydig cells of the postpubertal and adult testis (Fig. 3). was observed in small clusters of immature Leydig As for 3bHSD and P450c17 immunostaining in post- cells of prepubertal testis and intensity increased with pubertal testis, Leydig cells stained in tight, well-defined age. In addition, 3bHSD immunolabel was observed clusters, whereas in adult testis Leydig cells were stained in Sertoli cells within seminiferous tubules of pre- scattered throughout the interstitial space. When pubertal testes but was not detected in Sertoli cells of primary antisera were omitted, no staining was observed postpubertal and adult testes. The presence of 3bHSD (Fig. 3). Similarly, CYP19 (P450arom gene) mRNA in Leydig cells observed in the current study agrees expression was higher in adult testes compared with with the results reported by Hejmej & Bilinska (2008) prepubertal and postpubertal testes (Table 1). In addition, and suggests that Leydig cells are the main source of P450arom mRNA expression was higher in postpubertal de novo steroid synthesis in the testes. However, than prepubertal testes (Table 1). 3bHSD was also located inside the seminiferous tubule of prepubertal testis, indicating that seminiferous tubule may also be involved in androgen Discussion at some lower level. In our RT-qPCR experiments, This study examined the expression and localization of an increase by age was also observed, which suggests enzymes involved in sex steroid synthesis in prepu- that expression increases at the protein and the bertal, postpubertal, and adult equine testes. 3bHSD is transcriptional levels. www.reproduction-online.org Reproduction (2011) 141 841–848

Downloaded from Bioscientifica.com at 09/26/2021 10:20:34PM via free access 844 J Almeida and others

AB

CD

EF

Figure 2 P450c17 immunolabeling in prepuber- tal (A), postpubertal (C), and adult (E) testis with respective negative controls (B, D, and F). P450c17 immunolabeling was observed in small clusters of immature Leydig cells (bold arrows) of prepubertal testis and increased with age. 50.0 µm Immunolabel was not observed when primary antisera were omitted.

P450c17 enzyme characteristics are well known in (Raeside et al. 2006). In addition, CYP17 and HSD3B1 mammals (Miller 1988, Conley & Bird 1997). It is expression has been reported in Leydig cells and other expressed at high levels in classical steroidogenic tissues testicular cells, such as Sertoli cells and spermatogenic such as testis, , and of some species cells in several species including the monkey (Liang et al. as well as rodent, bovine, ovine, and porcine placenta. 1998), American black bear (Tsubota et al. 1997), P450c17 catalyzes two sequential reactions: the first is Japanese black bear (Okano et al. 2003), Hokkaido the 17a-hydroxylation of PREG and PROG to 17-OH- brown bear (Tsubota et al. 1993), and Japanese raccoon PREG and 17-OH-PROG respectively and the second is dog (Qiang et al. 2003). Our results suggest that the 17,20-lyase cleavage of 17-OH-PREG and 17-OH- androgen synthesis mainly occurs in Leydig cells of the PROG to DHEA and ADION respectively (Nakajin & equine testis and increases with age. Hall 1981, Zuber et al. 1986). In this study, P450c17 Androgens produced by Leydig cells and, possibly, immunolabel was observed in small clusters of immature Sertoli cells serve as substrates for estrogens production Leydig cells of prepubertal testis and immunolabel by P450arom. P450arom is present in the ER of intensity increased with age. P450c17 immunolabel numerous vertebrate tissues and has been the focus of wasalsoobservedintheseminiferoustubuleof studies in numerous species (Conley & Hinshelwood prepubertal testes, which decreased in the adult testis. 2001). According to Carreau et al. (1999) and Hess et al. In addition, CYP17 mRNA increased from prepubertal (2001), the primary source of estrogen is the Sertoli cell to adult testis, which is consistent with our immuno- in the immature testis and Leydig and germ cells in the histochemical observations. LH is the main regulator of mature testis. In the pig (Conley et al. 1996) and the testosterone biosynthesis by Leydig cells, and P450c17 human (Inkster et al. 1995), P450arom was reported to activity has been detected in Leydig cells of various be present only in Leydig cells, whereas in the rodent species such as the rat (O’Shaughnessy & Murphy (Carreau et al. 2007) its expression was observed in 1991), mouse (Nolan & Payne 1990), human (Hammar Sertoli cells during development and in Leydig cells in & Petersson 1986), goat (Weng et al. 2005), and boar the adult testis. In addition, Nitta et al. (1993)

Reproduction (2011) 141 841–848 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/26/2021 10:20:34PM via free access Expression of steroidogenic enzymes 845

AB

CD

EF

Figure 3 P450arom immunolabeling in prepu- bertal (A), postpubertal (C), and adult (E) testis with respective negative controls (B, D, and F). P450arom immunolabeling was absent or slightly observed in immature Leydig cells (bold arrows) of the prepubertal testis and increased with age. P450arom immunolabeling was not observed inside seminiferous tubules. Immuno- 50.0 µm label was not observed when primary antisera were omitted. demonstrated P450arom in germ cells of mice. Thus, accordance with those reported by Hess & Roser (2004), several distinct sites express that P450arom and estrogen in which postpubertal testes expressed P450arom inside synthesis seems to evolve with age in different species. In Leydig cells, we were not able to observe the age- the stallion, the gonads are the major source of estrogen. dependent shift they observed. According to Hess & Until 2 years of age, urinary and plasma levels of Roser (2004), P450arom immunolabel shifted from estrogen are very low and start to increase during the Leydig and Sertoli cells in prepubertal testis to only following years. Several studies have reported the Leydig cells in the postpubertal testis. In our experi- presence of P450arom in the equine testis (Eisenhauer ments, the range of age considered as prepubertal was et al. 1994, Almadhidi et al. 1995, Hess & Roser 2004) from 10 to 12 months of age, whereas that of Hess & and suggested an age-related shift in the cellular location Roser was from 3 to 7 months. However, their range for of P450arom from Leydig and Sertoli cells in the pubertal horses was from 12 to 18 months and they prepubertal testes to only Leydig cells in the postpubertal reported a slight degree of positive staining within the testes (Hess & Roser 2004). In this study, we demon- seminiferous tubules, which we did not observe. The strated very faint immunolabeling of P450arom in Leydig different antisera used may explain such differences, and cells of prepubertal testis with a significant increase in antisera specificity might be an issue to be considered postpubertal and adult testes. However, we did not when comparing both experiments. In this study, we observe P450arom immunolabel in Sertoli cells of detected immunolabel in Leydig cells only. Therefore, prepubertal, postpubertal, or adult testes. In accordance we suggest that Leydig cells are primarily responsible to our IHC data, CYP19 mRNA expression significantly for estrogen production in the equine testis and its increased between prepubertal, postpubertal, and adult expression increases with age. stages. The fact that P450arom was observed only in In conclusion, the data are consistent with the Leydig cells of postpubertal and adult testes agrees with expression of P450c17 and 3bHSD in Leydig cells of previous studies in the horse (Eisenhauer et al. 1994, prepubertal testes, which increased with age. CYP17 Almadhidi et al. 1995). Even though our findings are in and HSD3B1 mRNA expression also increased with age, www.reproduction-online.org Reproduction (2011) 141 841–848

Downloaded from Bioscientifica.com at 09/26/2021 10:20:34PM via free access 846 J Almeida and others

Table 2 Primer sequence and efficiency for real-time quantitative PCR of equine testis. mRNA Reference number Primer sequence Primer efficiency (%) CYP19 Horse genome # Forward: 50-CCACATCATGAAACACGATCA-30 101.4 AF031520.1 Reverse: 30-TACTGCAACCCAAATGTGCT-50 CYP17 GenBank accession # Forward: 50-GCATGCTGGACTTACTGATCC-30 98.8 D30688.1 Reverse: 30-CTGGGCCAGTGTTGTTATTG-50 HSD3B1 GenBank accession # Forward: 50-AGCAAATACCATGAGCACGA-30 110 D89666.1 Reverse: 30-TAACGTGGGCATCTTGTGAA-50 EqB2M Equine genome accession # Forward: 50-GATAGTTAAGTGGGATCGAGACCTCT-30 100 NM_001082502 Reverse: 30-GCACAATTTGGAACTAGTCAAATCC-50 which suggests an increase at the protein and transcrip- Medical School, Edinburgh, UK; Mapes et al. 1999), and tional levels. 3bHSD was also observed inside anti-human P450arom (1:1000, polyclonal rabbit, generously seminiferous tubules (presumptive Sertoli cells) of provided by Dr Nobuhiro Harada, Fujita Health University prepubertal testis and expression in the tubular compart- School of Medicine, Aichi, Japan; Conley et al. 1996, Walters ment disappeared in the postpubertal testis. P450arom et al. 2000). Antisera were diluted in PBS prior to was absent or observed at low levels in immature Leydig immunolabeling. cells of prepubertal testes but immunolabeling signi- After incubation with the primary antisera, slides were rinsed ficantly increased in intensity and together with mRNA for 5 min in PBS and incubated with a biotinylated second expression in the postpubertal and adult testis. We antisera (1:2000; goat anti-rabbit IgG) for 30 min prior to conclude from these data that Leydig cells are the main detection using the Vectastain ABC detection kit (Vectorlabs). cells responsible for androgen and estrogen production Slides were then counterstained with hematoxylin and mounted in aqueous mounting media (DakoCytomation, in the horse testis. However, Sertoli cells may also make Carpinteria, CA, USA). a minor contribution to androgen production. To determine the specificity of primary antisera labeling, slides were incubated with normal rabbit sera instead of the primary. Materials and Methods

During routine castrations, testes from prepubertal (10–12 Quantitative real time PCR months of age; nZ10), postpubertal (2 years of age; nZ5), and adult (3–9 years of age; nZ4) stallions were collected and RNA was extracted from a small amount of tissue (!30 mg) divided in two samples. One sample was fixed in buffered using Qiagen’s RNeasy Mini Kit. cDNA was generated using neutral formalin, embedded in paraffin, and sectioned at 5 mm Qiagen’s QuantiTect RT Kit, which has an integrated genomic for IHC. The other sample was snap frozen on dry ice and DNA removal step. Primers specific for the known sequence stored at K80 8C for mRNA isolation. Castrations were of equine CYP17, HSD3B1, and CYP19 were designed using performed in the Veterinary Medical Teaching Hospital at the Roche’s Universal Probe primer design website (Roche University of California, Davis, between January and May of Diagnostics, Indianapolis, IN, USA). CYP17 and HSD3B1 2008, 2009, and 2010. sequences were obtained from GenBank and CYP19 was obtained from the University of California Santa Cruz’s (UCSC) horse genome (Table 2). Equine b2-microglobulin sequence Immunohistochemistry was obtained from UCSC horse genome and was used as Fixed tissues were deparaffinized through CitriSolv (Fisher reference gene (Livak & Schmittgen 2001). Primers were used at Scientific, Pittsburgh, PA, USA), dehydrated through a graded 100 mM concentration and probes (8 bp with a fluorescent alcohol series (100, 95, and 70% ethanol), and endogenous (6-carboxy-fluorescein) label were used at 10 mM. Qiagen’s QuantiTect Probe PCR kit contained a HotStarTaq DNA peroxidases were quenched with 0.3% H2O2 in methanol for 30 min. Slides to be probed with P450c17 and 3bHSD primary polymerase, ROX passive reference dye, and a dNTP mix in an antisera were processed for antigen retrieval with antigen optimized buffer. The cycling conditions for qPCR were initially unmasking solution (Vectorlabs; Burlingame, CA, USA) for 15 min at 95 8C incubation, followed by 95 8C for 15 s, and 5 min at 93 8C. Slides to be probed for P450arom were not 60 8C for 60 s, repeated 40 times. Amplification was measured subjected to antigen retrieval and were washed with PBS using an ABI 7900 (Applied Biosystems, Foster City, CA, USA). immediately after endogenous peroxidase quenching. After rinsing in PBS, slides were blocked with normal rabbit sera for Statistical analyses 20 min at 20 8C. The sections were then incubated in humidified chambers overnight at 4 8C with the following For RT-qPCR analyses, the difference between threshold cycles primary antisera: anti-P450c17 (1:3000, rabbit polyclonal anti- (DCt) was calculated by subtracting the Ct value of the target bovine; Peterson et al. 2001), anti-3bHSD (1:1500, polyclonal gene from that of the reference gene and subjected to ANOVA rabbit antihuman, generously provided by Dr J Ian Mason, to generate DDCt values (Yuan et al. 2006; JMP ver 8; SAS, Cary, Centre for Reproductive Biology, University of Edinburgh NC, USA).

Reproduction (2011) 141 841–848 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/26/2021 10:20:34PM via free access Expression of steroidogenic enzymes 847

Declaration of interest Kwon S, Hess RA, Bunick D, Nitta H, Janulis L, Osawa Y & Bahr JM 1995 Rooster testicular germ cells and epididymal sperm contain P450 The authors declare that there is no conflict of interest that aromatase. Biology of Reproduction 53 1259–1264. (doi:10.1095/ could be perceived as prejudicing the impartiality of the biolreprod53.6.1259) research reported. Le Goascogne C, Sananes N, Eychenne B, Gouezou M, Baulieu EE & Robel P 1995 Androgen biosynthesis in the stomach: expression of cytochrome P450 17 alpha-hydroxylase/17,20-lyase messenger ribo- nucleic acid and protein, and of pregnenolone and Funding progesterone by parietal cells of the rat gastric mucosa. Endocrinology This study was supported by John P Hughes Endowment, 136 1744–1752. (doi:10.1210/en.136.4.1744) Levallet J, Bilinska B, Mittre H, Genissel C, Fresnel J & Carreau S 1998 UCDavis Center of Equine Health, CAPES and Fulbright. Expression and immunolocalization of functional cytochrome P450 aromatase in mature rat testicular cells. Biology of Reproduction 58 919–926. (doi:10.1095/biolreprod58.4.919) Liang JH, Sankai T, Yoshida T, Cho F & Yoshikawa Y 1998 Localization of References testosterone and 3beta-hydroxysteroid dehydrogenase/delta5-delta4- Abney TO 1999 The potential roles of estrogens in regulating Leydig cell isomerase in cynomolgus monkey (Macaca fascicularis) testes. development and function: a review. 64 610–617. (doi:10.1016/ Journal of Medical Primatology 27 10–14. S0039-128X(99)00041-0) Liang JH, Sankai T, Yoshida T, Cho F & Yoshikawa Y 1999 Localization of Almadhidi J, Seralini GE, Fresnel J, Silberzahn P & Gaillard JL 1995 immunoreactive testosterone and 3beta-hydroxysteroid dehydrogenease/ Immunohistochemical localization of cytochrome P450 aromatase in delta5-delta4 isomerase in cynomolgus monkey (Macaca fascicularis) equine gonads. Journal of Histochemistry and Cytochemistry 43 testes during postnatal development. Journal of Medical Primatology 28 571–577. (doi:10.1177/43.6.7769228) 62–66. Bilinska B, Lesniak M & Schmalz B 1997 Are ovine Leydig cells able Livak KJ & Schmittgen TD 2001 Analysis of relative gene expression data to aromatize androgens? Reproduction, Fertility, and Development 9 using real-time quantitative PCR and the 2(KDelta Delta C (T)) Method. 193–199. (doi:10.1071/R96038) Methods 25 402–408. (doi:10.1006/meth.2001.1262) Carreau S, Genissel C, Bilinska B & Levallet J 1999 Sources of oestrogen in Majdic G, Millar MR & Saunders PT 1995 Immunolocalisation of androgen the testis and reproductive tract of the male. International Journal of receptor to interstitial cells in fetal rat testes and to mesenchymal and Andrology 22 211–223. (doi:10.1046/j.1365-2605.1999.00172.x) epithelial cells of associated ducts. Journal of Endocrinology 147 Carreau S, Silandre D, Bourguiba S, Hamden K, Said L, Lambard S, 285–293. (doi:10.1677/joe.0.1470285) Galeraud-Denis I & Delalande C 2007 Estrogens and male reproduction: Mapes S, Corbin CJ, Tarantal A & Conley A 1999 The primate adrenal a new concept. Brazilian Journal of Medical and Biological Research 40 zona reticularis is defined by expression of cytochrome b5, 17alpha- 761–768. (doi:10.1590/S0100-879X2007000600003) hydroxylase/17,20-lyase cytochrome P450 (P450c17) and NADPH- Conley AJ & Bird IM 1997 The role of cytochrome P450 17 alpha- cytochrome P450 reductase (reductase) but not 3beta-hydroxysteroid hydroxylase and 3 beta-hydroxysteroid dehydrogenase in the integration dehydrogenase/delta5-4 isomerase (3beta-HSD). Journal of Clinical of gonadal and adrenal steroidogenesis via the delta 5 and delta 4 Endocrinology and Metabolism 84 3382–3385. (doi:10.1210/jc.84.9. pathways of steroidogenesis in mammals. Biology of Reproduction 56 3382) 789–799. (doi:10.1095/biolreprod56.4.789) Miller WL 1988 Molecular biology of synthesis. Endocrine Conley A & Hinshelwood M 2001 Mammalian . Reproduction Reviews 9 295–318. (doi:10.1210/edrv-9-3-295) 121 685–695. (doi:10.1530/rep.0.1210685) Conley AJ, Rainey WE & Mason JI 1994 Ontogeny of steroidogenic enzyme Nakajin S & Hall PF 1981 Microsomal cytochrome P-450 from neonatal pig expression in the porcine conceptus. Journal of Molecular Endocrinology testis. Purification and properties of a C21 steroid side-chain cleavage 12 155–165. (doi:10.1677/jme.0.0120155) system (17alpha-hydroxylase-C17,20 lyase). Journal of Biological Conley AJ, Corbin CJ, Hinshelwood MM, Liu Z, Simpson ER, Ford JJ & Chemistry 256 3871–3876. Harada N 1996 Functional aromatase expression in porcine adrenal Nitta H, Bunick D, Hess RA, Janulis L, Newton SC, Millette CF, Osawa Y, gland and testis. Biology of Reproduction 54 497–505. (doi:10.1095/ Shizuta Y, Toda K & Bahr JM 1993 Germ cells of the mouse testis express biolreprod54.2.497) P450 aromatase. Endocrinology 132 1396–1401. (doi:10.1210/en.132. Eisenhauer KM, McCue PM, Nayden DK, Osawa Y & Roser JF 1994 3.1396) Localization of aromatase in equine Leydig cells. Domestic Animal Nolan CJ & Payne AH 1990 Genotype at the P450scc locus determines Endocrinology 11 291–298. (doi:10.1016/0739-7240(94)90020-5) differences in the amount of P450scc protein and maximal testosterone Haider SG & Servos G 1998 Ultracytochemistry of 3beta-hydroxysteroid production in mouse Leydig cells. Molecular Endocrinology 4 dehydrogenase in Leydig cell precursors and vascular endothelial cells 1459–1464. (doi:10.1210/mend-4-10-1459) of the postnatal rat testis. Anatomy and Embryology 198 101–110. O’Donnell L, Robertson KM, Jones ME & Simpson ER 2001 Estrogen and (doi:10.1007/s004290050168) spermatogenesis. Endocrine Reviews 22 289–318. (doi:10.1210/er.22.3. Hammar M & Petersson F 1986 Testosterone production in vitro in human 289) testicular tissue. Andrologia 18 196–200. (doi:10.1111/j.1439-0272. O’Shaughnessy PJ & Murphy L 1991 Steroidogenic enzyme activity in the 1986.tb01761.x) rat testis following Leydig cell destruction by ethylene-1,2-dimethane- Hejmej A & Bilinska B 2008 The effects of cryptorchidism on the regulation sulphonate and during subsequent Leydig cell regeneration. Journal of of steroidogenesis and gap junctional communication in equine testes. Endocrinology 131 451–457. (doi:10.1677/joe.0.1310451) Endokrynologia Polska 59 112–118. Okano T, Murase T & Tsubota T 2003 Spermatogenesis, serum testosterone Hess MF & Roser JF 2004 Immunocytochemical localization of cytochrome levels and immunolocalization of steroidogenic enzymes in the wild P450 aromatase in the testis of prepubertal, pubertal, and postpubertal horses. male Japanese black bear (Ursus thibetanus japonicus). Journal of Theriogenology 61 293–299. (doi:10.1016/S0093-691X(03)00237-1) Veterinary Medical Science 65 1093–1099. (doi:10.1292/jvms.65.1093) Hess RA, Bunick D & Bahr J 2001 Oestrogen, its receptors and function Papadopoulos V, Carreau S, Szerman-Joly E, Drosdowsky MA, Dehennin L in the male reproductive tract – a review. Molecular and Cellular & Scholler R 1986 Rat testis 17beta-estradiol: identification by gas Endocrinology 178 29–38. (doi:10.1016/S0303-7207(01)00412-9) Inkster S, Yue W & Brodie A 1995 Human testicular aromatase: immuno- chromatography–mass spectrometry and age related cellular distri- cytochemical and biochemical studies. Journal of Clinical Endocrinology bution. Journal of Steroid Biochemistry 24 1211–1216. (doi:10.1016/ and Metabolism 80 1941–1947. (doi:10.1210/jc.80.6.1941) 0022-4731(86)90385-7) Keeney DS, Jenkins CM & Waterman MR 1995 Developmentally regulated Peterson JK, Moran F, Conley AJ & Bird IM 2001 Zonal expression of expression of adrenal 17alpha-hydroxylase cytochrome P450 in the mouse endothelial nitric oxide synthase in sheep and rhesus adrenal cortex. embryo. Endocrinology 136 4872–4879. (doi:10.1210/en.136.11.4872) Endocrinology 142 5351–5363. (doi:10.1210/en.142.12.5351) www.reproduction-online.org Reproduction (2011) 141 841–848

Downloaded from Bioscientifica.com at 09/26/2021 10:20:34PM via free access 848 J Almeida and others

Qiang W, Murase T & Tsubota T 2003 Seasonal changes in spermatogenesis Walters KW, Corbin CJ, Anderson GB, Roser JF & Conley AJ 2000 Tissue- and testicular steroidogenesis in wild male raccoon dogs (Nyctereutes specific localization of cytochrome P450 aromatase in the equine procynoides). Journal of Veterinary Medical Science 65 1087–1092. embryo by in situ hybridization and immunocytochemistry. Biology of (doi:10.1292/jvms.65.1087) Reproduction 62 1141–1145. (doi:10.1095/biolreprod62.5.1141) Raeside JI, Christie HL, Renaud RL & Sinclair PA 2006 The boar testis: the Wattenberg LW 1958 Microscopic histochemical demonstration of steroid- most versatile steroid producing organ known. Society of Reproduction 3 beta-ol dehydrogenase in tissue sections. Journal of Histochemistry and Fertility Supplement 62 85–97. and Cytochemistry 6 225–232. Sasano H, Mason JI & Sasano N 1989 Immunohistochemical analysis of Weng Q, Medan MS, Ren L, Watanabe G, Tsubota T & Taya K 2005 cytochrome P-450 17alpha-hydroxylase in pig adrenal cortex, testis and Immunolocalization of steroidogenic enzymes in the fetal, neonatal and ovary. Molecular and Cellular Endocrinology 62 197–202. (doi:10.1016/ adult testis of the Shiba goat. Experimental Animals 54 451–454. (doi:10. 0303-7207(89)90006-3) 1538/expanim.54.451) Tsubota T, Nitta H, Osawa Y, Mason JI, Kita I, Tiba T & Bahr JM 1993 Yuan JS, Reed A, Chen F & Stewart CN Jr 2006 Statistical analysis of Immunolocalization of steroidogenic enzymes, P450scc, 3beta-HSD, real-time PCR data. BMC Bioinformatics 7 85. (doi:10.1186/1471-2105- P450c17, and P450arom in the Hokkaido brown bear (Ursus arctos 7-85) yesoensis) testis. General and Comparative Endocrinology 92 439–444. Zuber MX, Simpson ER & Waterman MR 1986 Expression of (doi:10.1006/gcen.1993.1180) bovine 17alpha-hydroxylase cytochrome P-450 cDNA in nonsteroido- Tsubota T,Howell-Skalla L, Nitta H, Osawa Y,Mason JI, Meiers PG, Nelson RA genic (COS 1) cells. Science 234 1258–1261. (doi:10.1126/science. &BahrJM1997 Seasonal changes in spermatogenesis and testicular 3535074) steroidogenesis in the male black bear Ursus americanus. Journal of Reproduction and Fertility 109 21–27. (doi:10.1530/jrf.0.1090021) Vianello S, Waterman MR, Dalla Valle L & Colombo L 1997 Develop- Received 6 December 2010 mentally regulated expression and activity of 17alpha-hydroxylase/ C-17,20-lyase cytochrome P450 in rat liver. Endocrinology 138 First decision 5 January 2011 3166–3174. (doi:10.1210/en.138.8.3166) Accepted 7 February 2011

Reproduction (2011) 141 841–848 www.reproduction-online.org

Downloaded from Bioscientifica.com at 09/26/2021 10:20:34PM via free access