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365 Expression levels of Mullerian-inhibiting substance, GATA4 and 17-hydroxylase/17,20-lyase cytochrome P450 during embryonic gonadal development in two diverse breeds of swine

S A McCoard, T H Wise and J J Ford United States Department of Agriculture, Agricultural Research Service, US Meat Research Center, Clay Center, Nebraska 68933, USA (Requests for offprints should be addressed to S A McCoard who is now at Nutrition and Behaviour Group, AgResearch Limited, Private Bag 11008, Tennent Drive, Palmerston North, New Zealand; Email: [email protected])

Abstract Sexual differentiation and early embryonic/fetal advancing gestation, with greater levels of MIS and development is a tightly regulated process controlled by P450c17 in testes of MS compared with WC . numerous endocrine and molecular signals. These signals Organization of ovarian medullary cords and formation of ensure appropriate structural organization and subsequent nests was evident at similar ages in both breeds; development of and accessory organs. Substantial however, a greater number of MS compared with WC differences exist in adult reproductive characteristics in embryos exhibited signs of ovarian differentiation at 30 Meishan (MS) and White Composite (WC) pig breeds. dpc. In summary, despite breed differences in MIS and This study compared the timing of embryonic sexual P450 levels in the testis, which may be related to Sertoli c17 differentiation in MS and WC pigs. Embryos/fetuses were and Leydig function, the timing of testicular differen- evaluated on 26, 28, 30, 35, 40 and 50 days postcoitum tiation did not differ between breeds and is unlikely to (dpc). Gonadal differentiation was based on morphological impact reproductive performance in adult boars. In con- criteria and on localization of GATA4, Mullerian- trast, MS embryos exhibited advanced ovarian inhibiting substance (MIS) and 17-hydroxylase/17,20- differentiation compared with WC embryos which may lyase cytochrome P450 (P450c17). The timing of testicular be related to the earlier reproductive maturity observed in cord formation and functional differentiation of Sertoli this breed. and Leydig cells were similar between breeds. Levels of Journal of Endocrinology (2002) 175, 365–374 GATA4, MIS and P450c17 proteins increased with

Introduction Gonadogenesis is a tightly regulated process that involves co-ordinated expression of several developmen- In occidental pig breeds, genetically determined gonads tally expressed genes. Several genes are known to play begin to form on the ventromedial surfaces of the meso- important roles in gonadal development and sexual differ- nephroi around 20–21 days postcoitum (dpc) (Pelliniemi entiation including SRY (Koopman et al. 1990), SOX9 1975, McCoard et al. 2001a). Male sexual differentiation (Kent et al. 1996, Morais da Silva et al. 1996), Mullerian- occurs around 26 dpc (Pelliniemi 1975, Tung et al. 1984, inhibiting substance (MIS) (Munsterberg & Lovell-Badge Tung & Fritz 1987, McCoard et al. 2001a), characterized 1991), GATA4 (Heikinheimo et al. 1997, Viger et al. by the formation of testicular cords. Shortly thereafter, 1998, Ketola et al. 1999, McCoard et al. 2001a), steroido- Leydig cells of the testicular interstitium differentiate, genenic factor 1 (SF1) (Ikeda et al. 1994), DAX1 (Swain resulting in androgen-stimulated development of the male et al. 1996) and Wilms tumor factor-1 (WT1) (Pritchard- phenotype. Female sexual differentiation is initiated later Jones et al. 1990). In pigs, GATA4 is expressed prior to the in development (33 dpc), characterized by the forma- morphological differentiation of the bipotential gonads tion of medullary cords (McCoard et al. 2001a). By 40 dpc, (McCoard et al. 2001a), followed by the expression of abundant egg cell nests and well-organized medullary and SOX9, SF1 and WT1 in both (Parma et al. 1999). cortical regions of the are present (McCoard et al. Coincident with male sexual differentiation, Sertoli cells 2001a) and by 44 dpc the medullary cords have degener- begin to produce MIS transcripts and protein (Tran et al. ated, indicating the completion of ovarian differentiation 1977, Parma et al. 1999, McCoard et al. 2001a), initiating (Pelliniemi & Lauteala 1981). regression of the Mullerian ducts (Knebelmann et al. 1991,

Journal of Endocrinology (2002) 175, 365–374 Online version via http://www.endocrinology.org 0022–0795/02/0175–365  2002 Society for Endocrinology Printed in Great Britain

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Table 1 Total numbers of used and the proportion of embryonic/fetal gonads that exhibit signs of Sertoli and Leydig cell differentiation (male sexual differentiation) or the formation of ovarian medullary cords and formation of egg cell nests (female sexual differentiation)

Animals Males

Age (dpc) Litters Total fetuses MIS (%) P450c17 (%) MC (%) Nests (%) Breed WC 28 6 74 100 46 0 0 30 4 42 100 100 15a 0 35 6 43 100 100 100 0 40 6 71 100 100 100 100 50 8 59 100 100 100 100 MS 28 6 54 100 57 0 0 30 4 43 100 100 76a 0 35 5 47 100 100 100 0 40 3 31 100 100 100 83 50 2 25 100 100 100 100

aP<0·01, WC compared with MS. MIS (%): the proportion of embryonic/fetal gonads that produce MIS protein, a marker of Sertoli cell differentiation. P450c17 (%): the proportion of embryonic/fetal gonads that produce P450c17, a marker of Leydig cell differentiation. MC (%): the proportion of embryonic/fetal gonads that contain medullary cords (MC) indicative of the initiation of female sexual differentiation. Nests (%): the proportion of embryonic/fetal gonads that contain egg cell nests (nests) indicative of female sexual differentiation. Note: data for 26 dpc fetuses are not presented here. Despite initiation of male sexual differentiation in both breeds at this age, accurate identification of all males and females was not achievable at this age. These data are presented in the text in relation to all embryos irrespective of karyotype.

Behringer et al. 1994, Imbeaud et al. 1996, Mishina et al. objective of this study was therefore to utilize both 1996), thereby inhibiting development of the female duct morphological criteria and molecular markers of sexual system. MIS activity continues to increase in the testis differentiation and gonadal development to compare the from 27 to 33 dpc (Tran et al. 1977) suggesting a potential timing of sexual differentiation and subsequent gonadal role in testicular cord development. Thus, MIS is widely development in male and female MS and WC embryos. used as a functional marker of Sertoli cell differentiation. Profiles and levels of various gene products implicated in Subsequent to secretion of MIS, differentiated Leydig sexual differentiation and early gonadal development were cells produce several steroidogenic enzymes including examined using densitometric quantification. 17-hydroxylase/17,20-lyase cytochrome P450 (P450c17) (Conley et al. 1994, Greco & Payne 1994, Kaminski et al. 1999), which functions as an enzymatic catalyst for Materials and Methods testosterone synthesis (Moon et al. 1973, Lejeune et al. 1998), facilitating androgen-stimulated development of Sample collection the Wolffian ducts. Chinese Meishan (MS) boars reach earlier, but Embryos were collected from pure-bred MS and WC have smaller testes due to reduced Sertoli cell numbers and (YorkshireLandrace) sows (minimum of second parity) lower production than White Composite (WC) bred to MS and WC boars, respectively. A subset of WC boars (Okwun et al. 1996a,b, Lunstra et al. 1997). MS embryos was also used in an independent study (McCoard females achieve puberty at a younger age, and have a et al. 2001a). Estrus was checked daily and sows were higher rate and larger litters than WC females mated naturally on the first and second day of detected (Bolet et al. 1986, Ashworth et al. 1990, Christenson 1993, estrus, using different boars on each day. Day 0 corre- Faillace et al. 1994, Hunter et al. 1994). Differential sponds to the first day of mating. Embryos/fetuses were seminiferous tubule development and timing of Leydig collected on 26, 28, 30, 35, 40 and 50 dpc. Immediately cell differentiation have been reported in MS and WC after slaughter of the sows, body weight and crown– pigs (Kaminski et al. 1999), indicating that early develop- rump length (CRL) were recorded for each conceptus. mental events influence future reproductive performance. Embryonic length is a reliable indicator of gestational However, these conclusions were based on only one litter age (McCoard et al. 2001a) and was used to confirm of MS pigs per day of . Furthermore, in males, embryonic age. Embryos from 26 to 30 dpc were left marked differences in Sertoli cell development between intact, but paired mesonephroi with attached gonads were these two breeds are not evident until early neonatal life dissected from fetuses collected between 35 and 50 dpc. (McCoard et al. 2001b). Breed differences in the timing of The number of litters and the total number of embryos/ female development have not been investigated. The fetuses evaluated at each gestational age are presented in

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Figure 1 Brightfield images of immunohistochemically stained embryonic and early fetal porcine gonads from 26 to 50 dpc. The red color depicts positive staining and sections are lightly counterstained blue with hematoxylin. (A) Localization of MIS protein (arrows) in the early testicular cords of a male WC embryonic gonad at 26 dpc. (B) GATA4 protein localization in the testicular cords of a male WC embryonic gonad at 26 dpc. Note the darker staining of the differentiated Sertoli cells enabling the morphological identification of early testicular cords (arrows). (C) Localization of P450c17 in the interstitium (arrows) of an MS testis at 30 dpc. (D) MIS localization to the of Sertoli cells within the testicular cords (arrows) of a WC at 30 dpc. (E) GATA4 localization in Sertoli cell (sc) of testicular cords and Leydig cells (lc) of the interstitium at 40 dpc in a WC embryo. Note the absence of staining in germ cells (gc). (F) MIS localization in MS testicular cords (arrows) at 40 dpc. (G) GATA4 localization in cells of a WC ovary at 28 dpc. Note the absence of structural organization of the stromal tissue. (H) GATA4 localization in the somatic cells of an MS ovary at 35 dpc. Note the formation of early ovarian medullary cords (arrows) and the darker staining in the medullary cords. (I) GATA4 localization in an MS ovary at 50 dpc. Note the absence of staining in the germ cells and the formation of egg cell nests (arrow).

Table 1. Procedures for handling all animals in this study Sample preparation and immunohistochemistry complied with those specified in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and All samples were fixed in fresh 4% paraformaldehyde in Teaching (FASS 1999). Dulbeccos’s phosphate-buffered saline (PBS) overnight at www.endocrinology.org Journal of Endocrinology (2002) 175, 365–374

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4 C with gentle agitation (fixative was changed once parison of staining intensity between breeds, sexes and during incubation). Each sample was washed in PBS across ages for each protein evaluated. (21 h), dehydrated through graded ethanol (50, 70, 80, To correct for small fluctuations in either the specimen 90, 100%; 21 h each), cleared in xylene (21h; light source or in ambient room lighting, a stored back- Sigma, St Louis, MO, USA), infiltrated with paraffin wax ground image was used to standardize each captured (60 C; 41 h), and embedded in paraffin wax. All specimen image. Computers operate on a color scale from samples were subjected to the same processing conditions. 0 to 255 (0 is black and 255 is white), the inverse of Sections (5 µm) were made through the longitudinal conventional ‘absorption’ scales. Thus, the data are pre- axis of each embryo and mesonephroi/gonad. Cross- sented as the inverse of these arbitrary units to correspond sections were dried overnight onto glass slides at 37 C and to conventional ‘absorption’ scales. stained the following day. Sections were stained immuno- histochemically for GATA4 and MIS (Santa Cruz Statistical analysis Biotechnology, Santa Cruz, CA, USA) as previously ff described (McCoard et al. 2001a,b). The same immuno- Di erences between breeds and sexes in all measurements were tested using a mixed model procedure (SAS 1999). histochemical procedure was also used for the anti-P450c17 antibody (courtesy of Dr Anita Payne, Stanford University, For traits expressed in both sexes, the model included fixed ff Palo Alto, CA, USA) used at a dilution of 1:1000. Non- e ects of breed, and age, and two-way interactions, ff immune serum or absence of the primary antibodies was and the random e ect of litter nested within breed. Sex used to conclude that non-specific binding was not prob- was dropped from the model for those traits expressed in lematic. For each protein evaluated, all slides were subject only one sex. For body weight, data were transformed to to identical staining conditions. Slides were stored at room logarithms before statistical analyses to adjust for hetero- temperature in the dark until densitometric analysis. geneity of variances. Paired comparisons were made using To account for interassay variation in staining intensity, the Tukey–Kramer procedure. Data are presented as least testicular tissue from a boar at 105 dpc was selected as square means (LSM) and standard errors (body weight and control tissue. One section of this tissue was processed with CRL data) or inverse least square means (densitometric each assay irrespective of breed, sex, age or protein data). evaluated, and used to correct for interassay and intra-assay variation in staining intensity as described below. Results The presence of MIS protein in the cytoplasm of Sertoli Densitometric measurements cells (Fig. 1A) and formation of morphologically distin- Average density measurements were made using a guishable early testicular cords identified by GATA4 Bioquant Nova color imaging system (Bioquant Nova immunoreactivity (Fig. 1B, see McCoard et al. 2001a) Advanced Image Analysis 2000; R&M Biometrics, Inc.) indicated functional and morphological differentiation of and used to quantify the amount of protein present the testis by 26 dpc in both breeds. At this stage of for each gene examined using brightfield microscopy. development, 20–25% of all embryonic gonads examined Average density measures the average color value of a in both breeds contained MIS protein (Fig. 1A) and group of pixels and is the most common way to compare exhibited morphological signs of seminiferous tubule the amount of light-absorbing stain present in one object formation (Fig. 1B). GATA4 protein was present in the with others. Since average density depends only on color somatic cells of both the undifferentiated (absence of value, the average density value is independent of the testicular cords and MIS protein) and differentiated gonads size of the object under examination, allowing valid (Fig. 1B and E) but was never observed in germ cells or comparison of structures differing in size. endothelial cells of the vasculature. GATA4 was strongly One full cross-section from near the middle of each expressed in differentiated Sertoli cells enabling rapid gonad was used in these evaluations. Average density histomorphometric identification of male sexual differen- values were obtained for each protein in the entire tiation (Fig. 1B and E). GATA4 was also present in Leydig cross-section of each gonad. For each assay, immunoreac- cells of the interstitium (Fig. 1E), coelomic epithelial cells tive cells were defined by pre-setting a threshold value. In and some undifferentiated interstitial cells. Morphological this way, sections from each animal and the control slide and functional differentiation of testicular cords of all were treated in an identical manner. Due to interassay males, based on the presence of MIS and morphologically variation in staining intensity, slightly different threshold differentiated testicular cords, was evident by 28 dpc values were sometimes required to identify all immuno- (Table 1). In both breeds, P450c17 protein was first reactive cells. To account for interassay variation in identified in the cytoplasm of Leydig cells at 28 dpc (Table staining intensity and threshold values, the control density 1), indicative of their functional differentiation. By 30 dpc, value was subtracted from the treatment density values the presence of P450c17 and MIS in all testes irrespective within each assay. This approach enabled the valid com- of breed (Table 1) illustrated the presence of functionally

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Downloaded from Bioscientifica.com at 09/26/2021 10:04:04PM via free access Sexual differentiation in pigs · SAMCCOARD and others 369 differentiated Leydig cells (Fig. 1C) and Sertoli cells (Fig. Discussion 1D and 1F) respectively. Somatic cells of the contained GATA4 pro- This study describes the timing of sexual differentiation, tein (Fig. 1 G–I). Intense GATA4 immunoreactivity was spatial and temporal localization patterns and levels of observed in early ovarian medullary cords enabling rapid molecular markers associated with sexual differentiation identification of early signs of sexual differentiation of the and early gonad development in MS and WC conceptuses. ovary (Fig. 1H, see McCoard et al. 2001a). Intense Sexual differentiation is accompanied by changes in the GATA4 immunoreactivity of somatic cells and absence of structural organization of the gonad and onset of expression staining in germ cells enabled the rapid identification of of various developmentally expressed genes. egg cell nests (Fig. 1I). Based on the formation of ovarian Testicular cords were detected morphologically by 26 medullary cords, detected by histological examination of dpc in both breeds, consistent with previous reports in GATA4 immunoreactivity, the first signs of sexual differ- domestic pigs (Pelliniemi 1975, McCoard et al. 2001a). entiation of the female gonad were not evident until 30 Coincident with the formation of testicular cords, MIS dpc in both breeds (Table 1). At this stage of development, protein was observed in the cytoplasm of Sertoli cells in a greater proportion (P<0·001) of MS (76%) compared embryos of both breeds at 26 dpc, indicative of their with WC (15%) female embryos exhibited early morpho- functional differentiation (Tran et al. 1977). Well- logical features of sexual differentiation. Female embryos organized testicular cords and high levels of MIS protein at this stage of development were distinguished from testes were observed in large proportions of the gonads of both ff by the relatively unorganized structure of the ovarian tissue breeds by 28 dpc. Di erentiated Leydig cells (P450c17 (Fig. 1 G) and the absence of MIS and P450c17 protein. immunoreactive) were also detected in the testicular Ovarian medullary cords were present in all female gonads interstitium of both breeds from 28 dpc onwards, consist- of both breeds by 35 dpc (Table 1). By 40 dpc, all ovaries ent with previous reports (Moon et al. 1973, Pelliniemi from WC fetuses and 83% of MS fetal ovaries contained 1975, 1976). Collectively, these observations indicate egg cell nests indicative of sexual differentiation (Table 1). that the timing of differentiation of testicular cords and At this stage of development, distinctive regionalization of steroidogenic machinery of the testis do not differ between the ovaries into cortex and medulla regions was evident in MS and WC breeds. These observations contrast with the both breeds and all ovaries from both breeds contained report by Kaminski et al. (1999), where testicular cord numerous egg cell nests. Ovaries continued to increase in formation was delayed and P450c17 expression was not size up to 50 dpc, as did the total number of germ cells and observed until 34 dpc in MS embryos compared with 30 egg cell nests (Fig. 1I). dpc in WC (i.e. a 4-day delay). Discrepancies between Levels of MIS protein in testes increased rapidly from findings of the present study and the previous report are the onset of sexual differentiation (26 dpc) to 50 dpc in unlikely to be the result of differences in genetic back- both breeds (Fig. 2A). Testes from MS embryos/fetuses ground of MS pigs as they were derived from the same contained greater levels of MIS protein compared with base population. Rather, discrepancies are likely to result WC fetuses (Fig. 2A). GATA4 protein levels also from the number of MS embryos evaluated between 26 increased from the onset of sexual differentiation in both and 35 dpc. In the present study, 43–54 embryos from males and females (Fig. 2B), but did not differ between the four to six MS litters were evaluated at each gestational breeds or between the sexes at any age evaluated (Fig. 2B). age, and embryonic age was confirmed by CRL, a reliable Similar patterns of GATA4 and MIS protein in the testes indicator of gestational age (McCoard et al. 2001a). In the of both breeds were observed (Fig. 1A and B). Levels of previous study by Kaminski et al. (1999), only one MS P450c17 protein in the embryonic testes of both breeds litter (the number of embryos per litter was not reported) increased from the onset of Leydig cell differentiation (Fig. was evaluated at each age. 2C), and testes from MS embryos/fetuses contained Early signs of structural organization of fetal ovaries greater levels of P450c17 protein than testes from WC were detected in a greater proportion of MS compared embryos/fetuses (Fig. 2C). with WC ovaries at 30 dpc, indicating that MS embryos Body weight of both MS and WC embryos and fetuses initiate ovarian differentiation earlier (up to 5 days) than increased with advancing gestational age (Fig. 3A). A WC embryos. However, by 35 dpc, all ovaries from both divergence in body weight was observed between 40 and breeds contained early ovarian cords. Egg cell nests were 50 dpc in favor of WC compared with MS fetuses, but not detected until 40 dpc, at which time all WC ovaries breed differences in body weight were not observed at any and the vast majority of MS ovaries contained egg other stage evaluated. In both breeds, male fetuses had nests indicative of sexual differentiation (Byskov 1986). greater body weights compared with female fetuses (Fig. Organization of cortical cords and degeneration of 3A). CRL also increased with advancing age in both medullary cords characterizes final differentiation of the breeds, but did not differ between breeds (Fig. 3B). ovary (Pelliniemi & Lauteala 1981). Medullary cords were However, there was a tendency for males to have greater present in some ovaries of both breeds at 40 dpc but, by 50 CRL values than females in both breeds (Fig. 3B). dpc, medullary cords were absent from both breeds. www.endocrinology.org Journal of Endocrinology (2002) 175, 365–374

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Figure 2 (A) Densitometric values (arbitrary units) indicating the amount of Mullerian-inhibiting substance (MIS) protein in the testes of Meishan (MS) and White Composite (WC) embryos during late embryonic and early fetal life. NS, not significant. (B) Densitometric values (arbitrary units) indicating the amount of GATA4 protein in the gonads of male and female MS and WC embryos during late embryonic and early fetal life. (C) Densitometric values (arbitrary units) indicating the amount of 17-hydroxylase/17,20-lyase cytochrome P450 (P450c17) protein in the testes of male MS and WC embryos during late embryonic and early fetal life. *P<0·05.

Collectively, these observations illustrate that MS female morphological development in MS ovaries (S A McCoard, embryos initiate ovarian differentiation earlier than WC unpublished observations) and subsequent earlier onset of embryos which may be associated with advanced puberty and enhanced reproductive performance (Bolet

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this time and the presence of MIS and steroid production in the testis, support this concept in porcine gonadal differentiation. MIS and P450c17 are essential for normal male devel- opment. Increased MIS protein with advancing age is consistent with the requirement of high levels of MIS to cause regression of Mullerian ducts. Similarly, increased P450c17 with age is consistent with the requirement for testosterone production from the testis for maintenance of Wolffian ducts and subsequent development of male genitalia. In addition to the vital role of MIS on the Mullerian ducts, MIS has been implicated as a negative modulator of Leydig cell differentiation and function (Behringer et al. 1990, Mishina et al. 1996, Racine et al. 1998). However, the profiles of MIS and P450c17 protein in the present study do not support a role for MIS as a negative regulator of P450c17 expression in the embryonic pig. This is likely a reflection of the variety of species- and tissue-specific mechanisms of P450c17 gene regulation (Chung et al. 1987, DiBlasio et al. 1987, Mellon & Vaisse 1989, Mesiano et al. 1993, Staels et al. 1993, Givens et al. 1994, Greco & Payne 1994, Miller et al. 1997, Zhang & Mellon 1997). Profiles of both MIS and P450c17 protein exhibit similar patterns; however, MIS and P450c17 were both elevated in MS compared with WC testes. Observed differential secretory profiles were not associated with timing of initiation of Sertoli and Leydig cell differentia- tion. However, elevated production of MIS in MS Figure 3 (A) Body weight and (B) crown–rump length (CRL) testes may be related to MIS function, i.e. Mullerian (LSMS.E.) of MS and WC embryos during late embryonic and duct regression, which was not evaluated in this study. early fetal life. ***P<0·001. Similarly, elevated P450c17 in MS boars may be associated with increased testosterone synthesis and secretion from Leydig cells (Moon et al. 1973, Ford et al. 1980). Potential ff association of di erential production of MIS and P450c17 et al. 1986, Ashworth et al. 1990, Christenson 1993, with cell function, and relationship to future reproductive Faillace et al. 1994, Hunter et al. 1994). characteristics in these two diverse breeds of pigs remain to In males, sexual differentiation is an active process be elucidated. requiring initiation by SRY resulting in the differentiation The complex expression patterns of MIS in both the of Sertoli cells and Leydig cells which produce the testis and ovary (Donahoe et al. 1976, Takahashi et al. MIS and testosterone respectively. Mullerian 1986, Hirobe et al. 1992, Lee et al. 1994) indicate that this duct regression requires MIS, and maintenance of the gene is tightly regulated. The proximal promoter region of Wolffian duct system, later developing into the vas the MIS gene exhibits regions of evolutionary conservation deferens and epididymis, requires testosterone (Behringer in all mammalian species studied to date (Cate et al. 1986, et al. 1994). Early expression of MIS later followed by Guerrier et al. 1990, Munsterberg & Lovell-Badge 1991, P450c17 observed in male embryos in this study is consist- Haqq et al. 1992, Eusebe et al. 1996), including the pig ent with this developmental process. In contrast, female (Pilon et al. 1998). A variety of factors associated with sexual differentiation is a passive process whereby main- embryonic sexual differentiation are implicated in the tenance of the Mullerian duct, the precursor for Fallopian regulation of MIS expression (Haqq et al. 1994, Shen et al. tubes, and upper vagina, proceeds in the absence 1994, Giuili et al. 1997, Shimamura et al. 1997, Nachtigal of MIS, and degeneration of the Wolffian duct results et al. 1998, de Santa Barbara et al. 1998, Arango et al. because of lack of testosterone stimulation. The absence of 1999), including GATA4 (Viger et al. 1998, Tremblay & steroid production from fetal ovaries is likely to result from Viger 1999). GATA4 expression precedes that of MIS in the absence of steroidogenic enzymes in fetal ovaries, porcine Sertoli cells (McCoard et al. 2001a). The present ff including P450c17 (Greco & Payne 1994). Early signs of study indicates that, following testicular di erentiation ovarian differentiation at 30–35 dpc, coupled with the in the pig, GATA4 and MIS protein profiles are absence of both MIS and P450c17 in embryonic ovaries at similar, confirming temporal and spatial correlations in www.endocrinology.org Journal of Endocrinology (2002) 175, 365–374

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developmental expression patterns of GATA4 and MIS Christenson RK 1993 Ovulation rate and embryonic survival in previously observed (McCoard et al. 2001a), further Chinese Meishan and white crossbred pigs. Journal of Animal Science 71 supporting a role for GATA4 in porcine embryonic 3060–3066. Chung BC, Picado-Leonard J, Maniu M, Bienkowski M, Hall PF, gonadogenesis. Shively JE & Miller WL 1987 Cytochrome P450c17 (steroid 17 In conclusion, highly co-ordinated morphological alpha-hydroxylase/17,20 lyase): of human adrenal and testis changes and strict spatial and temporal patterns of MIS, cDNAs indicates the same gene is expressed in both tissues. PNAS 84 407–411. GATA4 and P450c17 production illustrate that embryonic sexual differentiation is a tightly regulated process in the Conley AJ, Rainey WE & Mason JI 1994 Ontogeny of steroidogenic enzyme expression in the porcine conceptus. Journal of Molecular pig. Male MS and WC embryos exhibited similar tem- Endocrinology 12 155–165. poral and spatial patterns of morphological development DiBlasio AM, Voutilainen R, Jaffe RB & Miller WL 1987 Hormonal indicating that the timing of differentiation of testicular regulation of messenger ribonucleic acids for P450 scc (cholesterol cords and steroidogenic machinery of the testis does not side-chain cleavage enzyme) and P450c17 (17 alpha- differ between these two breeds, and is unlikely to impact hydroxylase/17,20-lyase) in cultured human fetal adrenal cells. Journal of Clinical Endocrinology and Metabolism 65 170–175. future reproductive performance in pigs. However, while ff Donahoe PK, Ito Y, MarfatiaS&Hendren WH III 1976 The di erential expression of MIS and P450c17, two key production of Mullerian inhibiting substance by the fetal, neonatal proteins associated with sexual differentiation, did not and adult rat. of 15 329–334. influence the timing of sexual differentiation, implications Eusebe D, di Clemente N, Rey R, Pieau C, Vigier B, Josso N & for Sertoli and Leydig cell function warrant further inves- Picard JY 1996 Cloning and expression of the chick anti-Mullerian gene. Journal of Biological Chemistry 271 4798–4804. tigation. In contrast to males, female MS embryos exhib- ff ff Faillace LS, BiggsC&HunterMG1994Factors a ecting the age at ited advanced sexual di erentiation compared with WC onset of puberty, ovulation rate and time of ovulation in Chinese embryos, which may be related to future reproductive Meishan gilts. Journal of Reproduction and 100 353–357. characteristics, such as early onset of puberty observed for FASS 1999 Guide for the Care and Use of Agricultural Animals in this breed. Agricultural Research and Teaching, revised ed. 1. Savoy, IL: Federation of Animal Science Societies. Ford JJ, Christenson RK & Maurer RR 1980 Serum testosterone concentrations in embryonic and fetal pigs during sexual Acknowledgements differentiation. Biology of Reproduction 23 583–587. Giuili G, Shen WH & Ingraham HA 1997 The nuclear receptor SF-1 The authors thank Susan Hassler and Alan Kruger for mediates sexually dimorphic expression of Mullerian inhibiting their skilful technical assistance, the personnel of the substance, in vivo. Development 124 1799–1807. USMARC Swine Operations for care of animals, and Givens CR, Zhang P, Bair SR& Mellon SH 1994 Transcriptional Donna Griess for secretarial assistance. regulation of rat cytochrome P450c17 expression in mouse Leydig MA-10 and adrenal Y-1 cells: identification of a single protein that mediates both basal and cAMP-induced activities. DNA and Cell Biology 13 1087–1098. References Greco TL & Payne AH 1994 Ontogeny of expression of the genes for steroidogenic enzymes P450 side-chain cleavage, 3 beta-hydroxy- Arango NA, Lovell-BadgeR&BehringerRR1999 Targeted steroid dehydrogenase, P450 17 alpha-hydroxylase/ mutagenesis of the endogenous mouse Mis gene promoter: in vivo C17–20 lyase, and P450 aromatase in fetal mouse gonads. definition of genetic pathways of sexual development. Endocrinology 135 262–268. Cell 99 409–419. Guerrier D, Boussin L, Mader S, Josso N, KahnA&Picard JY 1990 Ashworth CJ, Haley CS, Aitken RP & Wilmut I 1990 Embryo Expression of the gene for anti-Mullerian hormone. Journal of survival and conceptus growth after reciprocal embryo transfer Reproduction and Fertility 88 695–706.  between Chinese Meishan and Landrace Large White gilts. Haqq C, Lee MM, Tizard R, Wysk M, DeMarinis J, Donahoe PK & Journal of Reproduction and Fertility 90 595–603. Cate RL 1992 Isolation of the rat gene for Mullerian inhibiting Behringer RR, Cate RL, Froelick GJ, Palmiter RD & Brinster RL substance. Genomics 12 665–669. 1990 Abnormal sexual development in transgenic mice chronically Haqq CM, King CY, Ukiyama E, Falsafi S, Haqq TN, Donahoe PK 345 expressing Mullerian inhibiting substance. Nature 167–170. & Weiss MA 1994 Molecular basis of mammalian sexual Behringer RR, Finegold MJ & Cate RL 1994 Mullerian-inhibiting determination: activation of Mullerian inhibiting substance gene 79 substance function during mammalian sexual development. Cell expression by SRY. Science 266 1494–1500. 415–425. Bolet G, Martina-Botte F, Locatelli P, Gruand J, Terqui M & Heikinheimo M, Ermolaeva M, Bielinska M, Rahman NA, Narita N, Berthelot F 1986 Components of prolificacy in hyperprolific Large Huhtaniemi IT, Tapanainen JS & Wilson DB 1997 Expression and White sows compared with the Meishan and Large White breeds. hormonal regulation of transcription factors GATA-4 and GATA-6 , Selection, 18 333–342. in the mouse ovary. Endocrinology 138 3505–3514. Byskov AG 1986 Differentiation of mammalian embryonic gonad. Hirobe S, He WW, Lee MM & Donahoe PK 1992 Mullerian Physiological Reviews 66 71–117. inhibiting substance messenger ribonucleic acid expression in Cate RL, Mattaliano RJ, Hession C, Tizard R, Farber NM, Cheung granulosa and Sertoli cells coincides with their mitotic activity. A, Ninfa EG, Frey AZ, Gash DJ, Chow EP, Fisher RA, Bertonis Endocrinology 131 854–862. JM, Torres G, Wallner BP, Ramachandran KL, Ragin RC, Hunter MG, Faillace LS & Picton HM 1994 Intrauterine and Manganaro DT, MacLaughlin DT & Donahoe PK 1986 Isolation of peripheral steroid concentrations and conceptus development in the bovine and human genes for Mullerian inhibiting substance and Meishan and Large White hybrid gilts. Reproduction, Fertility and expression of the human gene in animal cells. Cell 45 685–698. Development 6 783–789.

Journal of Endocrinology (2002) 175, 365–374 www.endocrinology.org

Downloaded from Bioscientifica.com at 09/26/2021 10:04:04PM via free access Sexual differentiation in pigs · SAMCCOARD and others 373

Ikeda Y, Shen W-H, Ingraham HA & Parker KL 1994 MunsterbergA&Lovel-Badge R 1991 Expression of the mouse Developmental expression of mouse steroidogenic factor-1, an anti-Müllerian hormone gene suggests a role in both male and essential regulator of the steroid hydroxylases. Molecular Endocrinology female sexual differentiation. Development 113 613–624. 8 654–662. Nachtigal MW, Hirokawa Y, Enyeart-VanHouten DL, Flanagan JN, Imbeaud S, Belville C, Messika-Zeitoun L, Rey R, di Clemente N, Hammer GD & Ingraham HA 1998 Wilms’ tumor 1 and Dax-1 JossoN&Picard JY 1996 A 27 base-pair deletion of the modulate the orphan nuclear receptor SF-1 in sex-specific gene anti-Mullerian type II receptor gene is the most common cause of expression. Cell 93 445–454. the persistent Mullerian duct syndrome. Human Molecular Genetics 5 Okwun OE, Igboeli G, Lunstra DD, Ford JJ & Johnson L 1996a 1269–1277. Testicular composition, number of A spermatogonia, Kaminski MA, Corbin CJ & Conley AJ 1999 Development and ratios, and number of in three different breeds of boars. differentiation of the interstitial and tubular compartments of fetal Journal of 17 301–309. porcine testes. Biology of Reproduction 60 119–127. Okwun OE, Igboeli G, Ford JJ, Lunstra DD & Johnson L 1996b Kent J, Wheatley SC, Andrews JE, Sinclair AH & Koopman P 1996 Number and function of Sertoli cells, number and yield of A male-specific role for SOX9 in vertebrate sex determination. spermatogonia, and daily sperm production in three breeds of boar. Development 122 2813–2822. Journal of Reproduction and Fertility 107 137–149. Ketola I, Rahman N, Toppari J, Belinska M, Porte-Tinge SB, Parma P, PailhouxE&CotinotC1999 Reverse transcription- Tapanainen JS, Huhtaniemi IT, Wilson DB & Heikinheimo M polymerase chain reaction analysis of genes involved in gonadal 1999 Expression and regulation of transcription factors GATA-4 and differentiation in pigs. Biology of Reproduction 61 741–748. GATA-6 in developing mouse testis. Endocrinology 140 1470–1480. Pelliniemi LJ 1975 Ultrastructure of the early ovary and testis in pig Knebelmann B, Boussin L, Guerrier D, Legeai L, Kahn A, Josso N & embryos. American Journal of Anatomy 144 89–111. Picard JY 1991 Anti-Mullerian hormone Bruxelles: a nonsense Pelliniemi LJ 1976 Ultrastructure of the indifferent gonad in male and mutation associated with the persistent Mullerian duct syndrome. female pig embryos. Tissue and Cell 8 163–174. PNAS 88 3767–3771. Pelliniemi LJ & Lauteala L 1981 Development of Koopman P, Munsterberg A, Capel B, VivianN&Lovell-Badge R in the embryonic gonad. Human Genetics 58 64–67. 1990 Expression of a candidate sex-determining gene during mouse Pilon N, Behdjani R, Daneau I, Lussier JG & Silversides DW 1998 testis differentiation. Nature 348 450–452. Porcine steroidogenic factor-1 gene (pSF-1) expression and analysis Lee MM, Gustafson ML, Ukiyama E, Donahoe PK, MacLaughlin of embryonic pig gonads during sexual differentiation. Endocrinology DT, WexlerM&Keeping HS 1994 Developmental changes in 139 3803–3812. Mullerian inhibiting substance in the cynomolgus monkey, Macaca Pritchard-Jones K, Fleming S, Davidson D, Bickmore W, Porteous D, fascicularis. Journal of Clinical Endocrinology and Metabolism 78 Gosden C, Bard J, Buckler A, Pelletier J, Housman D, van 615–621. HeyningenV&Hastie N 1990 The candidate Wilms’ tumour Lejeune H, Sanchez P & Saez JM 1998 Enhancement of long-term gene is involved in genitourinary development. Nature 346 testosterone secretion and steroidogenic enzyme expression in 194–197. human Leydig cells by co-culture with human Sertoli cell-enriched preparations. International Journal of Andrology 21 129–140. Racine C, Rey R, Forest MG, Louis F, Ferre A, Huhtaniemi I, Josso N & di Clemente N 1998 Receptors for anti-Mullerian hormone Lunstra DD, Ford JJ, KlindJ&WiseTH1997 Physiology of the ff Meishan boar. Journal of Reproduction and Fertility 52 (Suppl) on Leydig cells are responsible for its e ects on steroidogenesis and ff 95 181–193. cell di erentiation. PNAS 594–599. McCoard SA, Wise TH, Fahrenkrug SC & Ford JJ 2001a Temporal de Santa Barbara P, Bonneaud N, Boizet B, Desclozeaux M, Moniot and spatial localization patterns of Gata4 during porcine B, Sudbeck P, Scherer G, PoulatF&Berta P 1998 Direct gonadogenesis. Biology of Reproduction 65 366–374. interaction of SRY-related protein SOX9 and steroidogenic factor 1 McCoard SA, Lunstra DD, Wise TH & Ford JJ 2001b Specific regulates transcription of the human anti-Mullerian hormone gene. 18 staining of Sertoli cell nuclei and evaluation of Sertoli cell number Molecular and Cellular Biology 6653–6665. and proliferative activity in Meishan and White Composite boars SAS 1999 SAS User’s Guide: Statistics, edn. 8. Cary, NC: SAS Inst during the neonatal period. Biology of Reproduction 64 689–695. Inc. Mellon SH & Vaisse C 1989 cAMP regulates P450 scc gene Shen WH, Moore CC, Ikeda Y, Parker KL & Ingraham HA 1994 expression by a cycloheximide-insensitive mechanism in cultured Nuclear receptor steroidogenic factor 1 regulates the Mullerian mouse Leydig MA-10 cells. PNAS 86 7775–7779. inhibiting substance gene: a link to the sex determination cascade. 77 Mesiano S, Coulter CL & Jaffe RB 1993 Localization of cytochrome Cell 651–661. P450 cholesterol side-chain cleavage, cytochrome P450 17 Shimamura R, Fraizer GC, Trapman J, Lau YC & Saunders GF 1997 alpha-hydroxylase/17, 20-lyase, and 3 beta-hydroxysteroid The Wilms’ tumor gene WT1 can regulate genes involved in sex ff dehydrogenase isomerase steroidogenic enzymes in human and determination and di erentiation: SRY, Mullerian-inhibiting rhesus monkey fetal adrenal glands: reappraisal of functional substance, and the androgen receptor. Clinical Cancer Research 3 zonation. Journal of Clinical Endocrinology and Metabolism 77 2571–2580. 1184–1189. Staels B, Hum DW & Miller WL 1993 Regulation of steroidogenesis Miller WL, Auchus RJ & Geller DH 1997 The regulation of 17,20 in NCI-H295 cells: a cellular model of the human fetal adrenal. lyase activity. Steroids 62 133–142. Molecular Endocrinology 7 423–433. Mishina Y, Rey R, Finegold MJ, Matzuk MM, Josso N, Cate RL & Swain A, Zanaria E, Hacker A, Lovell-BadgerR&Camerino G 1996 Behringer RR 1996 Genetic analysis of the Mullerian-inhibiting Mouse Dax-1 expression is consistent with a role in sex substance signal transduction pathway in mammalian sexual determination as well as adrenal and hypothalamus function. Nature differentiation. Genes and Development 10 2577–2587. Genetics 12 404–409. Moon YS, Hardy MH & Raeside JI 1973 The early differentiation of Takahashi M, Hayashi M, Manganaro TF & Donahoe PK 1986 The the testis and interstitial cells in the fetal pig, and its duplication in ontogeny of mullerian inhibiting substance in granulosa cells of the organ culture. American Journal of Anatomy 138 253–267. bovine . Biology of Reproduction 35 447–453. Morais da Silva S, Hacker A, Harley V, Goodfellow P, Swain A & Tran D, Muesy-DessoleN&JossoN1977Anti-Mullerian hormone Lovell-Badge R 1996 Sox9 expression during gonadal development is a functional marker of foetal Sertoli cells. Nature 269 411–412. implies a conserved role for the gene in testis differentiation in Tremblay JJ & Viger RS 1999 Transcription factor GATA-4 enhances and . Nature Genetics 14 62–68. Mullerian inhibiting substance gene transcription through a direct www.endocrinology.org Journal of Endocrinology (2002) 175, 365–374

Downloaded from Bioscientifica.com at 09/26/2021 10:04:04PM via free access 374 SAMCCOARD and others · Sexual differentiation in pigs

interaction with the nuclear receptor SF-1. Molecular Endocrinology mouse gonadal development and is a potent activator of the 13 1388–1401. Mullerian inhibiting substance promoter. Development 125 Tung PS & Fritz IB 1987 Morphogenetic restructuring and formation 2665–2675. of basement membranes by Sertoli cells and testis peritubular cells ZhangP&Mellon SH 1997 Multiple orphan nuclear receptors in coculture: inhibition of the morphogenetic cascade by cyclic converge to regulate rat P450c17 gene transcription: novel AMP derivates and by blocking direct cell contact. Developmental mechanisms for orphan nuclear receptor action. Molecular Biology 20 139–153. Endocrinology 11 891–904. Tung PS, Skinner MK & Fritz IB 1984 Cooperativity between Sertoli cells and peritubular myoid cells in the formation of the basal lamina in the seminiferous tubule. Annals of the New York Academy of Sciences 438 435–446. Viger RS, Mertineit C, Trasler JM & Nemer M 1998 Transcription Received 26 June 2002 factor GATA-4 is expressed in a sexually dimorphic pattern during Accepted 10 July 2002

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