REPRODUCTIONREVIEW

Cryptorchidism in common eutherian mammals

R P Amann and D N R Veeramachaneni Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado 80523-1683, USA Correspondence should be addressed to R P Amann; Email: [email protected]

Abstract Cryptorchidism is failure of one or both testes to descend into the scrotum. Primary fault lies in the testis. We provide a unifying cross-species interpretation of testis descent and urge the use of precise terminology. After differentiation, a testis is relocated to the scrotum in three sequential phases: abdominal translocation, holding a testis near the internal inguinal ring as the abdominal cavity expands away, along with slight downward migration; transinguinal migration, moving a cauda epididymidis and testis through the abdominal wall; and inguinoscrotal migration, moving a s.c. cauda epididymidis and testis to the bottom of the scrotum. The gubernaculum enlarges under stimulation of insulin-like peptide 3, to anchor the testis in place during gradual abdominal translocation. Concurrently, testosterone masculinizes the genitofemoral nerve. Cylindrical downward growth of the peritoneal lining into the gubernaculum forms the vaginal process, cremaster muscle(s) develop within the gubernaculum, and the cranial suspensory ligament regresses (testosterone not obligatory for latter). Transinguinal migration of a testis is rapid, apparently mediated by intra-abdominal pressure. Testosterone is not obligatory for correct inguinoscrotal migration of testes. However, normally testosterone stimulates growth of the vaginal process, secretion of calcitonin gene-related peptide by the genitofemoral nerve to provide directional guidance to the gubernaculum, and then regression of the gubernaculum and constriction of the inguinal canal. Cryptorchidism is more common in companion animals, pigs, or humans (2–12%) than in cattle or sheep (%1%). Laboratory animals rarely are cryptorchid. In respect to non-scrotal locations, abdominal testes predominate in cats, dogs, and horses. Inguinal testes predominate in rabbits, are common in horses, and occasionally are found in cats and dogs. S.c. testes are found in cattle, cats and dogs, but are most common in humans. Reproduction (2007) 133 541–561

Introduction inbreeding or minimized by culling affected males and all siblings. This is too simplistic. Approximately 25 years Cryptorchidism is failure of one or both testes to ago, the notion of a single locus gene problem gave way ‘descend’ into the scrotum at the time normal for the to acceptance of a polygenic recessive model, based species of interest, before or shortly after birth. on relatively small studies with pigs (Sittmann & Obviously, detection is postnatal. This problem was described by de Graaf in 1668 (Jocelyn & Setchell 1972), Woodhouse 1977, Rothschild et al. 1988); dogs (Cox in respect to humans, dogs and rams. De Graaf cited et al. 1978, Nielen et al. 2001); and also for men (Czeizel publications from 1582 and 1618, and presented et al. 1981). Unfortunately, techniques of modern personal observations of testicles located in the abdomi- molecular genetics have not been applied to sub-human nal cavity, near the kidneys, or in the groin area rather species with a sufficient incidence of cryptorchidism to than in the scrotum. Both uni- and bilateral cryptorchid- justify a study of gene abnormalities (e.g. cryptorchid ism were recognized. These early reports could have deer (from an unusual locale; Veeramachaneni et al. been interpreted (but were not) as evidence that 2006), dogs, horses, or pigs). Nevertheless, it is unlikely cryptorchidism is phenotypic evidence of two or more that sequence changes in 1–4 genes account for most diseases, because undescended testes are not always cases of cryptorchidism in common animals. This located at the same non-scrotal site. conclusion is based on comprehensive analyses for Until recently, the general perception was that hundreds of men; no single gene, considered to be cryptorchidism was a single disease with moderate involved in regulation of testicular descent, is aberrant in heritability, incomplete penetrance, expressed only in O10% of cryptorchid men (Ferlin et al. 2003, Roh et al. males (sex-specific expression), and concentrated by 2003, Klonisch et al. 2004, Garolla et al. 2005, Yoshida

q 2007 Society for Reproduction and Fertility DOI: 10.1530/REP-06-0272 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/01/2021 08:57:07PM via free access 542 R P Amann and D N R Veeramachaneni et al. 2005). It is now accepted that there is a multiplicity Early in development, a thin fold of peritoneum, the of causes for cryptorchidism (Hutson et al. 1997, mesonephric sheath, supports the mesonephros and Klonisch et al. 2004, Hutson & Hasthorpe 2005), provides the cranial suspensory ligament, which con- including genetic, epigenetic, and environmental nects the cranial tip of the future gonad to the components. dorsocranial abdominal cavity (Fig. 1, top). Assume a Cryptorchidism should be viewed as the ‘tip of an XY male with genes necessary to drive male develop- iceberg’, providing early and facile phenotypic detection ment, rather than default female development. Early in of testicular disease, which after puberty might be embryogenesis, primordial germ cells (PGCs) migrate evidenced as other phenotypic defects. These include from the hind gut to the gonadal ridge, on the quantitative and/or qualitative defects in spermatogen- ventromedial aspect of the mesonephros. Then esis, or tumors found long after abnormal differentiation mesenchymal cells, probably from the neighboring of anlage for germ, Sertoli, Leydig, or stromal cells. coelomic epithelium, move into the developing gonad, Although all of these defects might not be detected in a proliferate, and surround the PGCs; in the male they given individual, each is considered to be part of a differentiate into fetal Sertoli cells and secrete anti- testicular dysgenesis syndrome (TDS), and possibly all Mu¨llerian hormone (AMH), which induces demise of the have a common underlying cause from improper paramesonephric (Mu¨llerian) duct. The AMH also might development of fetal testes. This topic has received affect Leydig cell function. An early consequence of the much attention in the past 10–15 years (Skakkebaek interaction of fetal Sertoli cells with PGCs is that the et al. 1998, 2001, Rajpert-De Meyts 2006, Sharpe 2006). latter are prevented from entering meiosis, although The concept of a TDS does not exclude other causative proliferation and differentiation can occur. At least in mechanisms for cryptorchidism, such as pituitary failure. mice, PGCs do not enter meiosis because retinoic acid is Importantly, non-cryptorchidism does not guarantee not available within the seminiferous cords of a fetal freedom from other elements of TDS. testis (Koubova et al. 2006). A need to better understand the process of testicular Shortly after arrival offuture fetal Sertoli cells, other cells descent on a comparative basis arose as we explored an from the mesonephros follow to stimulate proliferation of unusually high incidence of cryptorchidism in a unique fetal Sertoli cells, organize nests of fetal Sertoli cells along population of deer (Veeramachaneni et al. 2006; with PGCs into seminiferous cords (Fig. 1,center;Sharpe summarized later herein), while we separately observed 2006), cooperate with fetal Sertoli cells to produce a basal disparate manifestations of cryptorchidism in rabbits lamina, and apparently give rise to the peritubular myoid exposed to chemicals or environmental pollutants (Veeramachaneni 2006). We found no recent unifying cells of an adult seminiferous tubule. More or less review of testis descent and pathophysiology of both concurrently, mesenchymal cells (probably from coelomic cryptorchid and non-cryptorchid testes covering epithelium, but mesonephric origin not excluded) migrate domesticated, companion, and laboratory animals into spaces among the seminiferous cords and differentiate together with humans. Hence, we undertook preparation into fetal Leydig cells. The interval between entrance of of a comprehensive review. The evolving manuscript PGCs into the indifferent gonad (not shown) through % was unwieldy, so it was split. Herein, we summarize formation of seminiferous cords requires 7 days in non- comparative information on early testis differentiation, rodent species, and differentiation of the gonad to a ! structures, and processes involved in testicular descent, functional testis (Fig. 1, bottom) is completed in 14 days timing of testicular descent, incidence and nature of after PGCs arrived in the gonad. Although much growth cryptorchidism, and why the problem probably will not and refinement of function occurs later, differentiation of a be eliminated. A separate review (in preparation) will gonad to a testis (Fig. 1, center) producing hormones and consider how exposure to certain environmental agents growth factors is completed at approximately gestational might result in cryptorchidism and, for a subset of agents, day (GD): 14, mouse; 16, rat; 22, rabbit; 34, dog; 35, horse; tumors of the testis or male reproductive tract. 36, pig; 42, bull; and 56, human. Within 2–3 days after arrival, fetal Leydig cells achieve maximum production of testosterone, and probably Formation of a testis insulin-like peptide 3 (Insl3). Initially, testosterone is Brief review of testis formation is essential to understand produced constitutively or under autocrine/paracrine how dysgenesis at this time could lead to seemingly control in rodents (El-Gehani et al. 1998, Pakarinen et al. diverse abnormalities, such as cryptorchidism, abnormal 2002), or possibly with stimulation of human chorionic spermatogenesis, tumors of the testis or excurrent duct gonadotropin (hCG) entering from maternal blood in system, or aplasia of male ducts. We augment excellent humans (Themmen & Huhtaniemi 2000), but later reviews focused on humans (Rajpert-De Meyts 2006) and luteinizing hormone (LH) and gonadotrophin-releasing rodents (Sharpe 2006), with information for other hormone (GnRH) come into play to regulate the process. common animals (Patten 1948, Gier & Marion 1969, As the testis continues to differentiate and grow, adult 1970, Bergin et al. 1970, Wensing & Colenbrander 1986). Leydig cells continue to produce Insl3 and testosterone.

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Figure 1 Structures involved in testis descent in a typical mammal; upper and lower sketches in lateral view and none in true scale. Upper, indifferent gonad: The gonadal ridge forms on ventromedial surface of mesonephros, suspended in folds of peritoneum leading to near the diaphragm (cranial suspensory ligament) and the inguinal area (gubernaculum). The gubernaculum grows out from mesenchymal cells within the abdominal musculature. The area of fusion between the gubernaculum and the external aspect of the mesonephric duct becomes the site of the future cauda epididymis. Center, testis formation: After primordial germ cells (PGCs) arrive in the gonad, and assuming genetic drive from a Y-chromosome, the PGCs are surrounded by fetal Sertoli cells and formed into nests, which then are organized into cords by fetal peritubular cells; fetal Leydig cells occupy inter-cord spaces. This is an early fetal testis. Because the 4 cell types intercommunicate via paracrine factors and hormones, abnormal function of 1 cell type early in fetal development likely affects the others and might irrevocably change gonocytes and eventually spermatogonia, and/or adult Sertoli, Leydig, and peritubular cells. Similarly, an exogenous agent affecting differentiation or programming of one cell type could permanently affect the others. Lower: The testis usurps the suspensory ligaments as the mesonephros degenerates, the peritoneal lining starts to evert as a vaginal process within the outer limits of the portion of the gubernaculum within the abdominal wall (this happens later in rodents and rabbits), fetal AMH drives regression of the paramesonephric duct (Mhllerian duct; not shown), and fetal testosterone drives the mesonephric duct to differentiate as the epididymal plus deferent duct and masculinization of the genitofemoral nerve (not shown). A little later, the accessory sex glands (not shown) also will develop from the mesonephric duct, but this requires dihydrotestosterone rather than testosterone. www.reproduction-online.org Reproduction (2007) 133 541–561

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Describing cryptorchidism ‘inguinoscrotal testis descent’, and consider testis descent to involve two phases rather than three phases In preparing this review, it became obvious that the topic as proposed herein. suffered from inappropriate and/or imprecise nomen- Accepting that there are three general locations for clature, which hindered cross-species comparisons, non-scrotal testes, and that testis descent involves three understanding of regulatory mechanisms, and interpret- phases, it follows that cryptorchidism reflects mani- ing actions of exogenous agents. Hence, we used festation of at least three prenatal diseases. These are: a) standard anatomical nomenclature (Schaller 1992, failure to initiate and complete abdominal testis International Committee 2005), and defined, and translocation; b) failure to initiate and complete suggested adoption of ‘process terms’ universally transinguinal migration of a testis; and c) failure to applicable to companion, food-producing, or wild initiate and complete inguinoscrotal migration of a testis. animals; rodents or rabbits; and humans. We have Causation of one of these three phenotypes is complex. taken the liberty of reinterpreting some conclusions in We will consider only the most likely causes of terminal older publications with the benefit of recent information, failure, namely insufficiency and timeliness of: Insl3, without discounting the underlying observations. Central intra-abdominal pressure or reduction of testis size, or in our review were publications describing numerous testosterone. dissections of fetuses, including those of: cattle (Gier & The term ‘testicular descent’ is typically used, but Marion 1969, 1970, Edwards et al. 2003); dog (Wensing ‘translocation’ probably is more descriptive of what 1968, Gier & Marion 1969); horse (Bergin et al. 1970); happens during the first phase; the absolute distance human (Hutson et al. 1990, 1997); pigs (Backhouse & between a testis and scrotal area changes little (see Butler 1960, Backhouse 1964, Wensing & Colenbrander below); the fetus grows away from the inguinal area, and 1986, Wensing 1988); rabbit (Rajfer 1980, Elder et al. the testes ‘stay put’ as the kidney is repositioned 1982, van der Schoot 1993, van der Schoot & Elger (Wensing 1968, Shono et al. 1994a). The term 1993); and mouse/rat (Wensing 1986, Wensing & ‘migration’ describes both movement of the testis Colenbrander 1986, van der Schoot & Elger 1993, van through the abdominal wall and also the separate der Schoot 1996, Shono et al. 1994a, 1994b; Hutson quest of the testis for the bottom of the scrotum, which et al. 1997, Lam et al. 1998, Hrabovszky et al. 2002). can be rather distant from the external inguinal ring. By definition, cryptorchidism refers to a postnatal With complete abdominal retention, both the testis phenotype. If one or both testes are not in the scrotum, and cauda epididymidis remained in the abdominal where are they? Usually non-scrotal testes are in one of cavity, with the testis near the kidney or part-way to the three general locations: abdominal cavity, inguinal canal internal inguinal ring and with the cauda epididymidis (canalicular), or s.c. (outside the abdominal wall). Many not juxtapositioned to the testis yet within the abdominal tabulations combine inguinal and s.c. locations under a cavity; the vaginal process had started evagination from single descriptor. However, there is no doubt that testes the abdominal wall. With incomplete abdominal are found within the inguinal canal in humans cryptorchidism (Stickle & Fessler 1978, Genetzky (Beltran-Brown & Villegas-Alvarez 1988, Rozanski & 1984), the cauda epididymidis had entered the inguinal Bloom 1995), horses (Rodgerson & Hanson 1997), and canal, but the testis remained within the abdominal rabbits (Veeramachaneni, unpublished). cavity, relatively close to the internal inguinal ring. Since cryptorchidism is failure of testis descent, An inguinal testis is within the canicular space limited location of a testis and not size, development, or by the internal and external inguinal rings. Ideally, molecular biology of associated structures (e.g. guber- position would be precisely defined (Beltran-Brown & naculum) should be the prime consideration in deciding Villegas-Alvarez 1988), and this would seem especially if the process involves two, three, or more phases. Non- important for horses since the inguinal canal might be scrotal testes are found in one of three general locations 10 cm long. A s.c. testis usually is found in the femoral (abdominal, inguinal, or s.c.), so it is logical that three triangle, but ectopia of the vaginal process might place phases are involved in the process of testis descent. the testis at some distance or near a malformed These are: a) abdominal testis translocation, specifically scrotum. Imprecision in describing testis location typifies retention near the neck of the developing bladder as the literature on mice or rats administered an agent, which abdominal cavity enlarges followed by slight testis might affect testis descent, and the uninformative relocation to the future internal inguinal ring; b) ‘ectopic testis’ (i.e. abnormal location of testis), which transinguinal migration of a testis, moving a cauda is often used to describe location of a testis not within a epididymidis and testis through the abdominal wall; and normal scrotum or abdominal cavity. Wolf et al. (2000) c) inguinoscrotal migration of a testis, from a s.c. provide an example of an adequate description. location outside the inguinal canal to correct final Categorizing s.c. testes as inguinal, vice versa, or position in the bottom of the scrotum. Most authors inguinoscrotal is common in cat, dog, horse and human have combined movement of a testis through the literature. This is inappropriate, and for stallions the abdominal wall and final migration to the scrotum as separate classification of inguinal and s.c. testes had

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Downloaded from Bioscientifica.com at 10/01/2021 08:57:07PM via free access Understanding cryptorchidism 545 been advocated by Genetzky (1984). Regardless, stallion peritoneum covering the gonad evolves to a mesorchium testes rarely are s.c. (Cox et al. 1979, Rodgerson & suspending the gonad dorsally from the mesonephros, Hanson 1997), but rather are within the inguinal canal and as the cranial suspensory ligament (Fig. 1), which per se. In humans, however, the majority of undes- blends into the diaphragmatic ligament supporting the cended testes apparently are ‘located in the groin’ or mesonephros. The cranial suspensory ligament is ‘near the neck of the scrotum or just outside the external sexually dimorphic, becoming substantial in females, inguinal ring’ (Hutson et al. 1992, 1997); i.e. s.c. Since it but not in male fetuses because it regresses during a is imprecise to attribute both conditions to failures of critical time window (Gier & Marion 1970, van der ‘inguinoscrotal testis descent’ and different regulatory Schoot & Emmen 1996, Hutson et al. 1997). Caudally, mechanisms are apparently involved, we use the term the peritoneum around the gonad continues as the thin ‘transinguinal migration’ for the former and restrict the caudal mesonephric sheath, which extends to the term ‘inguinoscrotal migration’ to the latter. extreme caudal end of the coelom (abdominal cavity). Since, there are two testes and at least three non- Early in development, the gubernaculum originates scrotal locations, a given cryptorchid male should be from mesenchymal cells among muscle fibers of the placed in one of six, if not 8–10, categories defined by a abdominal wall, grows under the peritoneal lining, and 2!3–4 matrix (2 sides, 3–4 combinations of testis and soon dominates the caudal mesonephric sheath. Thus, cauda epididymidis locations). Such complete infor- the gubernaculum extends from within the abdominal mation is rare. We have not found a data base pertaining wall, under (ventral to) the mesonephric duct with which to domestic, companion, or laboratory animals that it fuses (where the cauda epididymidis later transitions to provides information in adequate detail. The situation is the deferent duct), and connects to the testis. further complicated because the age at examination can affect what is found. This is especially important in species where testes typically reach a scrotal location What is the gubernaculum? between 10 days before birth and 14 days after birth The Latin word gubernaculum pertains to a ‘helm’ or a (horse, human, and pig; only then does the inguinal structure, which guides and was first applied to a canal constrict) or 3–20 days after birth (mouse, rat, and reproductive structure by Hunter (1762) because he rabbit; inguinal canal never constricts). Further, testes in thought that it guided the testis to the scrotum. Later, an inguinal location at first examination might later be he slightly modified his original description and wrote positioned permanently in the scrotum (late descent), (Hunter 1786)‘. which at present I shall call the and occasionally a scrotal testis might later be retracted ligament, or gubernaculum testis, because it connects permanently into the inguinal canal (‘retractile testis’ in the testis with the scrotum, and seems to direct its course human literature). Such migration is more common in through the rings of the abdominal muscles . it is horses, humans, or pigs than in cattle or sheep. certainly vascular and fibrous, and the fibers run in the direction of the ligament itself, which is covered by the fibers of the cremaster or musculus testis, placed Structures involved in testis descent immediately behind the peritoneum.’ Clearly, Hunter The same structures are involved in testicular descent in stated that the cremaster muscle covers the guber- common mammals (Fig. 1, bottom). Most important are naculum and, hence, he considered them as separate the testis formed from an undifferentiated gonad; structures. epididymal and deferent ducts formed from the Hunter (1762, 1786; cited text available on-line) mesonephric duct; mesonephros which degenerates; recognized that the morphology of the cremaster muscle metanephros which becomes the kidney; cranial differed among species, and considered it to originate suspensory ligament of testis (cephalic ligament); from the internal oblique muscle of the abdominal wall. gubernaculum (see below); vaginal process an evagi- The cremaster muscles are striated, and innervated by nation of the peritoneum (membrane lining abdominal the genitofemoral nerve. We now know that the cavity); cremaster muscle(s), which differs in shape gubernaculum has collagen fibers, is rich in hyaluronic among species; genitofemoral sensory nerve, with L1 acid and glycosaminoglycans, and its cells proliferate to L2 ganglia; and scrotum. There are marked species during expansion and include some myoblasts. Hunter differences with respect to relative timing of testis described (1762, 1786) what now is termed the vaginal descent, but the only substantial difference in structure process as a U-shaped evagination of peritoneum into, or process between most animals and rodents or rabbits and later through, the abdominal wall around the is with respect to transinguinal testis migration. Because gubernaculum. Hence, he probably considered the there are far more similarities than differences, we think vaginal process and gubernaculum as separate a unitary approach has merit. structures. The mesonephros, mesonephric duct leading to the As summarized previously, Hunter (1762, 1786) cloaca, and later the gonad, are within thin folds of considered the gubernaculum as a ligament and peritoneum. 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Downloaded from Bioscientifica.com at 10/01/2021 08:57:07PM via free access 546 R P Amann and D N R Veeramachaneni process. However, van der Schoot (1996) argued that In most animals, the vaginal process is formed by the gubernaculum be used as an encompassing term to parietal peritoneum invading the underlying guber- include the gubernaculum per se and also the vaginal naculum within the abdominal wall (Figs 2B and 3, process and cremaster muscles; in our opinion contrary to top). The evagination starts shortly after formation of a Hunter. In most reports on rodents or rabbits, the testis, and takes the shape of an incomplete cylinder cremaster muscles, but not the vaginal process, are (incomplete because of a reflection continuous with the considered part of the ‘gubernacular cone’, sometimes mesonephric sheath ultimately forming the mesorchium referred to as the gubernaculum without distinction supporting the testis and deferent duct). The vaginal between mesenchymal and muscular elements. In reports process divides the gubernacular bulb into three areas: pertaining to non-rodent species, distinction between the proper, central to the cylindrical vaginal process and gubernaculum and cremaster muscle(s) is typical. continuous with the gubernacular cord; vaginal, Distinction between the gubernaculum and cremaster concentric and outside the vaginal process; and muscle(s) is not a mere semantic problem. Failure to infravaginal, cup-shaped and between the invading make the distinction prevents proper description of peritoneum and distal tip. Downward invasion of the species differences in embryology or anatomy (e.g. vaginal process, from the developing internal inguinal ring, mouse or rat versus bull, horse, human, or pig) or through the gubernacular bulb continues after trans- association of observed defects to possible etiological inguinal testis migration, and extends into the developing factors. We propose universal adoption of the term extra-abdominal gubernaculum. The genitofemoral nerve ‘gubernaculum’ as excluding the cremaster muscles or (not shown) is carried downward with the gubernaculum the vaginal process, although both penetrate the and innervates the cremaster muscle. In rodents and gubernaculum as it enlarges during the process of testis rabbits, initial evagination of the vaginal process is descent. The term ‘gubernacular–cremaster complex’ is apparent just before reshaping of the gubernacular– cremaster complex; the latter is central to transinguinal proposed because it is more descriptive than ‘guber- testis migration. This difference among species in nacular cone’ favored by van der Schoot (1993, 1996). transinguinal testis migration is discussed later. Distinct use of the term ‘gubernacular–cremaster A striated cremaster muscle(s) is formed by myoblasts, complex’ facilitates consideration of structure–function migrating from the muscles of the abdominal wall and/or relationships and cross-species comparisons. We hope differentiating from mesenchymal cells of the guber- others will be precise, use clearly defined terms, and naculum. In any case, the cremaster muscle(s) invades adopt this terminology if they are not already using it. the vaginal portion of the gubernaculum (refer Fig. 2 in The gubernaculum originates, in the inguinal area, as Backhouse & Butler 1960). The cremaster muscle is mesenchymal cells among fibers of the oblique muscles strip-like in companion and food-producing animals or of the abdominal wall (Backhouse & Butler 1960, Gier & humans, located on the lateral aspect of the developing Marion 1969, 1970, Wensing 1986, 1988, Wensing & vaginal process. Colenbrander 1986, van der Schoot 1993, 1996). Soon In rodents or rabbits, two cremaster muscles the gubernaculum is evident as a broad-based bulge in develop as concentric and conspicuous layers the lower abdomen with a papilla invading the caudal encompassing the proper portion of the gubernacular mesonephric sheath (which is continuous with the lining bulb (Fig. 2F). They are continuous with the inner of the abdominal cavity). A narrow portion of the oblique and transverse muscles of the abdominal wall gubernaculum soon dominates the remainder of the (Wensing 1986, van der Schoot 1993, 1996, van der caudal mesonephric sheath and contacts the meso- Schoot & Elger 1993, Shono et al. 1994b). This results nephric duct and testis (Fig. 2A and D). These often are in paired ‘gubernacular–cremaster complexes’ (Fig. 3, described as a gubernacular cord plus a gubernacular bottom). The gubernacular–cremaster complex bulb, and they have functional differences. The portion includes the intra-abdominal gubernaculum and both of the gubernaculum initially within the abdominal wall cremaster muscles, but excludes the thin connection is knob-like and gelatinous with collagen fibers. (gubernacular cord) extending to the testis, apparently The upward-bulging papilla of gubernaculum is devoid of muscle cells, and the extra-abdominal transitory in many animals (compare images for dog gubernaculum. The gubernacular bulb and cremaster and bull in Fig. 2); the major (proper) portion of the muscle(s) have different roles during testis descent and gubernacular bulb seems to ‘settle’ into the abdominal later in adults. wall, accommodated by the peritoneal covering. In rodents and rabbits, the major portion of the guber- nacular bulb retains a conspicuous elongated-cone Process of testis descent shape (Fig. 2E and F) until just before transinguinal testis Although there are good descriptions of morphologic migration. In any case, the gubernacular bulb grows and changes during testis descent in common animals, there extends through the abdominal wall into the s.c. tissue. is a paucity of information on regulation of testis descent, This is the extra-abdominal portion of the gubernaculum. or agents disrupting the process, other than experiments

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Figure 2 Early development of the gubernaculum in dog, bull, and rat. (A): early in development in typical animals, the gubunacular bulb (GB) has a conical shape, protrudes into the abdominal cavity, and is continuous with the gubernacular cord (GC). (B) and (C): increasingly the GB is surrounded by the abdominal wall and the peritoneal lining grows downward as a vaginal process (VP). The internal end of the inguinal canal (IC) forms. (D): in the rat, mouse, or rabbit, the gubernacular bulb (GB) first becomes evident shortly before GD 16 in rat (75% of gestation). In the rat, as in the dog or bull (A-C), the portion of the gubernaculum protruding into the abdominal cavity has a conical shape; however this shape is maintained throughout gestation in rodents (birth on GD 21–22). (E) and (F): concentric cremaster muscles (CM) overlay the mesenchymal core of the gubernacular bulb (GB), forming a gubernacular-cremaster complex. (F): the muscle layer thickens gradually and the mesenchymal core increases in size and eventually extends further below the plane of the abdominal wall. Magnifications differ and are unknown. (A–C) are from Biology of Reproduction 1(Suppl 1), p 16, 1969, and used with permission from Elsevier. (D–F) are from Anatomical Record 236, pp 401–403, 1993, and used with permission from Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. All figures relabeled, and some rotated. with rodents and observations of humans. In our provide an anchor (Gier & Marion 1970, Edwards et al. synthesis, we summarize what is known from model 2003). Presumably the above changes along with species, augmented with observations on larger animals. fetal growth exert continuous tension on the testis, via We urge study of detailed reviews (Hutson et al. 1992, the gubernacular cord, while the cranial suspensory 1997, Heyns & Hutson 1995, Ong et al. 2005) and ligament weakens. Consequently, the testis is retained in especially Klonisch et al. (2004). Also refer Jost (1953) the inguinal region during migration of other structures and van der Schoot & Emmen (1996). (e.g. kidney) cranially. In cattle, the gubernaculum has developed sufficiently by GD 50 so that evagination of the peritoneum has Abdominal testis translocation started to form the vaginal process. Then between GD The endpoint is a testis positioned near the internal 62–65 and 90–96, the processes described previously inguinal ring, often with the cauda epididymidis just are completed (Fig. 4). As abdominal testis translocation within the inguinal canal. The process of abdominal proceeds, the testis increases in size (especially in the testis translocation is one avoiding cranial displacement stallion), accompanied by increased secretion of rather than substantial movement. The testis is anchored regulatory molecules. In most species (e.g. cattle, and by the cranial suspensory ligament and the guber- horse), the vaginal process is carried downward as the naculum (Fig. 1). Initially, the gubernaculum is short gubernaculum grows. and thin. The gubernaculum gradually expands and Abdominal testis translocation is accomplished with invades deeper into the abdominal musculature (Fig. 4). little change in the distance between a testis and the The extra-abdominal gubernaculum increases substan- scrotal area, although the extra-abdominal portion of the tially in size, by both cell division and swelling, to gubernaculum becomes longer (Fig. 5A; other data for www.reproduction-online.org Reproduction (2007) 133 541–561

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Figure 3 Comparison of testis descent in a typical mammal, rodent, or rabbit (upper, pig; lower, rat). Left-most drawings illustrate status early during abdominal translocation of testes. Other views depict intermediate stages continuing through transinguinal migration of the testis. In all animals, the gubernaculum grows out from the abdominal wall as well as downward, and the vaginal process is formed by invasion of the peritoneal lining. The gubernaculum has a gubernacular cord and bulb; the latter has 2 regions, shown for the rat as: d, intra-abdominal; and e, extra-abdominal. In all species, the gubernacular bulb later is divided by the invading vaginal process into 3 portions: a, proper; b, vaginal; and c, infravaginal. In most animals, the gubernacular bulb and vaginal process extend deep into the abdominal wall early during abdominal translocation of a testis (e.g. GD 60 pig), and the cremaster muscle grows downward through the extra-abdominal gubernaculum, as a relatively narrow strip on 1 side of the vaginal process. Abdominal testis translocation is completed around GD 100 in the pig. In a rodent or rabbit, starting around GD 16 in the rat, the cremaster muscle envelops the intra-abdominal portion of the gubernacular bulb, forming an extensive dome-like “gubernacular-cremaster complex”. In these species, down growth of the vaginal process does not occur until just before the end of abdominal translocation, around day 21–22. In pigs, transinguinal migration of testes occurs around GD 103–108, by movement through an inguinal canal distended by the proper portion (a) of the gubernaculum. In rodents and rabbits, intermediate stages depict intussusception of the cremaster muscles around the proper portion of the gubernaculum (e.g. day 23–24 rat, equals PND 2–4), which occurs postnatally in rodents, followed by straightening of the muscles to complete transinguinal testis migrationas the vaginal process grows. See text for details. For each species, the lower right drawing illustrates status at end of transinguinal migration (rat) or late during inguinoscrotal migration (pig) of testes. Drawings depict structures, and are not to scale within or among drawings. Drawings evolved from Elder et al. (1982), Wensing (1986, 1988), and Shono et al. (1994a), modified on basis of published plates and a consensus of literature. cattle and pigs in Wensing & Colenbrander 1986), and becomes O2.5 cm by GD 95. Maximum distance the fetus grows away from this area. For cattle, the between a testis and the future scrotum is at initiation distance between the internal inguinal ring and testis of transinguinal migration, at GD 95–100 in. remains approximately 1 cm until transinguinal Similarly in rodents and rabbits, the testis is held near migration (e.g. after GD 90 in Fig. 5A), whereas the the neck of the bladder, by the gubernaculum, as the distance between the internal inguinal ring and kidney abdominal cavity enlarges (Fig. 5B; Shono et al. 1994a).

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Figure 4 Abdominal testis translocation in a typical mammal is initiated shortly after testis differentiation and initial secretion of signaling molecules (see Fig. 1, center); approximately GD 43 in a bull fetus. By GD 62, formation of the vaginal process is conspicuous, the mesonephros is gone, and the testis is retained near the internal inguinal ring as the abdominal cavity enlarges and the metanephros (future kidney) is moved away from the testis and inguinal ring. Tension to hold the testis near the inguinal ring is provided by the gubernaculum. Based on data for model species, abdominal translo- cation is dependent on action of Insl3 peptide to stimulate gubernacular expansion. By GD 96, the testis is at the entrance to the Figure 5 Relative position of a testis, and other structures, during testis inguinal canal (i.e. internal inguinal ring) and typically the future cauda descent. (A): distance between the testis and internal inguinal ring epididymidis (not shown) is within the internal inguinal ring. Based on changes little in a bull fetus, whereas distance between the internal and redrawn from Gier & Marion (1970); also Wensing & Colenbrander inguinal ring and scrotum increases until initiation of transinguinal (1986) and Edwards et al. (2003). migration of the testis around GD 95. Development of the gubernaculum (purple) is primarily in the below the internal inguinal ring, in gubenacular By GD 16–17 in rats, GD 17 in mice, or GD 18–21 in bulb, initially to serve as an anchor to facilitate abdominal testis rabbits, the gubernacular–cremaster complex has translocation through tension on the gubernacular cord. Later diameter of the gubernacular bulb increases greatly and it distends the inguinal canal formed and assumed a conical or cylindrical shape to facilitate transinguinal testis migration. Distance between the testis and protruding into the abdominal cavity, from the femoral kidney increases greatly. Based on and redrawn from Edwards et al. triangle (Figs 2 and 3; Elder et al. 1982, van der Schoot (2003). Wensing (1968) provided similar data for bull and boar fetuses. 1993, van der Schoot & Elger 1993, Shono et al. 1996). (B): positions of the testis and ovary in fetal rats. Relative to the neck of the The gubernacular cord has shortened and the testes soon bladder, there is no substantial change in location of the testes after they form on GD w15 through GD 19, although the kidneys (not shown), and are below the neck of the bladder (Fig. 5B). By the end of the ovaries in a female fetus, are steadily moved cranially.After GD 19, the abdominal testis translocation, the gubernacular cord testes are caudal to the bladder neck although still within the abdominal has regressed, bringing the cauda epididymidis (already cavity. Abdominal translocation of a testis is completed around the time of attached to the future vaginal process by virtue of birth. Approximately 2–4 days after birth, transinguinal migration occurs attachment to the mesonephric sheath) against the and the gubernacular-cremaster structures and testes are positioned cremaster muscle or the intra-abdominal gubernaculum subcutaneously outside the abdominal cavity. Thereafter, 7–10 days are required for inguinoscrotal migration of testes to their final scrotal per se (Elder et al. 1982, Shono et al. 1996). position. Based on data in Shono et al. (1994a). Although rarely mentioned, inguinal canals apparently develop on postnatal day (PND) 1–2 in mice (Shono et al. 1996), prior to PND 5 in rats (Shono expanded, much like what had happened in a dog or et al. 1994b), and by GD 28 in rabbits (van der Schoot bull by GD 32–34 or 45–52. Further, the developing 1993). Given that an inguinal canal must be formed by vaginal process would initiate segregation of the vaginal cylindrical downward growth of the peritoneal lining, and proper portions of the gubernacular bulb and this means that the base of the gubernacular–cremaster demarcate the upper limit of the infravaginal guberna- complex had been repositioned slightly below the plane culum (Fig. 3, bottom). In both rodents and rabbits, of the abdominal wall as the rodent’s abdomen weight of the intra-abdominal gubernacular–cremaster www.reproduction-online.org Reproduction (2007) 133 541–561

Downloaded from Bioscientifica.com at 10/01/2021 08:57:07PM via free access 550 R P Amann and D N R Veeramachaneni complex increases five-fold from PND 0–6. Length of fetus or pup is growing far more rapidly than the gestation ranges over several days (10–15%), and in gubernacular–cremaster complex, which does not need many rat studies pups are removed surgically on GD 20 to ‘evert’ to relocate the testis (Lam et al. 1998). As noted so that PND 1 or 2 might be prenatal in another study. previously, the extra-abdominal gubernaculum extends Taking the end of abdominal translocation as position- into the inguinal canal or through the canal into s.c. ing of the testis close to the inguinal ring, and not change tissue (Fig. 7D, also refer to Fig. 8 in Elder et al. 1982) in the gubernaculum or birth as the endpoint, it is (Rajfer 1980, Elder et al. 1982, Shono et al. 1996, 2004). evident that the first phase of testis descent is completed Near PND 2 in rabbits (Rajfer 1980, Elder et al. 1982) around PND 0–1 in mice (Shono et al. 1994a, 1996)or or PND 4–8 in rats (Lam et al. 1998), the cremaster PND 4–5 in rats (Wensing 1986, Shono et al. 1994b). muscle assumes a serpentine or ill-defined appearance, Transinguinal migration of testes could not begin before with a diameter (across the intra-abdominal gubernacu- these ages. lum) greater than that of the testis. With a relatively large In all species, including rodents and rabbits, at the end portion of gubernaculum protruding along the external of abdominal testis translocation, the testis is positioned surface of the abdominal wall, reduction of the proper near the internal inguinal ring, the cauda epididymidis is within the inguinal canal (or poised to enter), the gubernaculum and vaginal process extend below the newly formed/forming inguinal canal (relatively short distances in rodents and rabbits), and the gubernaculum has both intra- and extra-abdominal regions (Fig. 3). In many species, this situation is maintained for some time, like a ‘pause’ between two separate processes. The genitofemoral nerve has been masculinized by the action of testosterone (Goh et al. 1994). The main force holding the testis low in the abdominal cavity is a considerably expanded gubernaculum.

Transinguinal testis migration The endpoint is a testis, and epididymis, located just external to the inguinal canal or plane of the abdominal wall. During the pause before actual transinguinal migration, the gubernacular bulb enlarges greatly (refer Fig. 7 in Gier & Marion 1970; also Figs 3 and 5) and dilates the inguinal canal to allow passage of the testis preceded by the cauda epididymidis. To accommodate transinguinal migration of the testis, the cranial suspen- sory ligament remains only as a thin sheet, and structures contributing to the future spermatic cord lengthen substantially. In due course, the testis decreases in absolute size and as the gubernacular bulb distends the inguinal canal sufficiently and the testis rapidly moves through (Figs 3, 6 and 7). The reduction in testis size is Figure 6 Transinguinal migration of testes in a typical mammal is substantial, especially in horse fetuses, so it can pass initiated after a pause following abdominal testis translocation and through the inguinal canal. Incompatibility between expansive development of the gubernacular bulb to dilate the inguinal testis size and diameter of the canal is considered by canal; the vaginal process and gubernaculum extend deep into the scrotum. By GD 95–102 in a bull fetus, the testis is entering the inguinal some, to be a contributing factor to abdominal canal (GD 100 in illustration). The actual start of transinguinal testis cryptorchidism in stallions. migration varies among males of a species, but actual migration Transinguinal testis migration is slightly different in through the canal apparently takes only 1–3 days as testes rarely are rodents and rabbits, for reasons presumably related to found within the inguinal canal in normal timed fetuses. Based on data substantial development of the dome-shaped cremaster for model species, massive expansion of the vaginal process later muscle. Dogma is that the gubernacular–cremaster necessary for inguinoscrotal migration of a testis might be facilitated by complex ‘everts’ as a herniation through the abdominal testosterone (or a metabolite thereof, e.g. dihydrotestosterone), perhaps acting on the genitofemoral nerve masculinized much earlier. By GD wall. We present a redistilled interpretation (Fig. 3, 105, the testis has passed through the external inguinal ring. bottom) of what happens (Elder et al. 1982, Wensing Inguinoscrotal migration (not shown) relocates the testis to the neck of 1986, van der Schoot & Elger 1993, Shono et al. 1994b, the scrotum (approximately GD 120 in bull) and to its final scrotal 2004, Lam et al. 1998). Central is the feature that the location by day 130. Based on and redrawn from Gier & Marion (1970).

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Actual passage of testes through the inguinal canal is thought to be rapid; a few days at most even in a large mammal. The gubernaculum per se probably has a passive role other than dilation of the inguinal canal (Wensing & Colenbrander 1986) and anchoring the cauda epidi- dymidis with attached testis. The gubernacular cord might shorten slightly. The main forces moving a testis through the inguinal canal are thought to be downward pressure of viscera and peritoneal fluid on the testis (harbored within the gubernacular bulb), expansion of the vaginal process, and growth of the abdomen.

Inguinoscrotal testis migration The endpoint is a testis, and epididymis, positioned normally in a scrotum typical of the species. Inguino- scrotal migration of a testis, from below the external inguinal ring to the final scrotal location, requires extension of the gubernacular bulb and enclosed vaginal process to the bottom of the scrotum, while the gubernacular cord does not elongate. In some species, the extra-abdominal gubernaculum might extend part- way into the scrotal folds well before transinguinal testis migration (see above), but because of fetal growth both gubernaculum and vaginal process must grow in the Figure 7 Locations of structures before, during, and after transinguinal proper direction to reach the bottom of the scrotal sac. In migration of testes. (A) gubernaculum and testis (T) in a GD 112 human rodents and rabbits the extra-abdominal gubernaculum, towards the end of abdominal migration of testis. The gubernacular with vaginal process, extend a relatively short distance bulb (G) protrudes below the inguinal canal although the cauda subcutaneously when transinguinal testis migration is epididymidis is not yet in the inguinal canal to position the testis against completed; they are not into the scrotum. In all species, the internal inguinal ring. (B) in a GD 120 bull, the gubernacular bulb extension of the vaginal process and gubernaculum over and testis have passed through the inguinal canal (Ic), and the gubernaculum extends deep into the scrotum although it is not attached a substantial distance from the external inguinal ring is thereto. (C) in PND 1 rat, the caudae epididymidis and testes (T) remain required to allow the gubernaculum to bring the cauda within the abdominal cavity and a short gubernacular cord (GC) epididymidis and testis to their proper locations. remains. Abdominal translocation of the testes is completed, but Inguinoscrotal testis migration involves growth of the transinguinal migration has not started. (D) on PND 7, the right testis gubernacular bulb and enclosed vaginal process in the (RT) is poised for transinguinal migration and the left testis (LT) has proper direction to the bottom of the scrotum. Over time, completed transinguinal migration and is outside the abdominal wall; its gubernacular bulb (GB) is directed towards the scrotum (Sc). The the gubernaculum regresses to attach the external surface actual tissue changes associated with transinguinal testis migration in a of the vaginal process, as the parietal vaginal tunic, to the rodent or rabbit probably are as in lower Fig. 3. Magnifications differ scrotal wall (via formation of scrotal ligament) and the and are unknown. From The Testis Volume 1, Gier HT & Marion GB, internal face to the cauda epididymidis (via formation of Development of the mammalian testis, pp 18, 22, 1970, and used with ligament of tail of epididymis). A remnant of the permission from Elsevier. All figures relabeled. gubernacular cord attaches the cauda epididymidis to the testis (proper ligament of testis). Except in rodents and intra-abdominal portion of gubernaculum, and growth of rabbits, the inguinal canal usually constricts to a very the pup, intussusception of the cylinder of cremaster narrow passageway precluding herniation of viscera. As muscle occurs (Elder et al. 1982) and, as it unfolds, it detailed below, the primary stimulus for inguinoscrotal assumes a U-shape around the cauda epididymidis and migration of a testis is chemoattractant signals received by testis (Fig. 3, bottom). This brings the cauda epididymidis the tip of the gubernacular bulb. and testis through the newly formed inguinal canal and outside the abdominal wall, with the cauda epididymidis still associated with the infravaginal portion of the Scrotal development and directional guidance gubernaculum. At this point, transinguinal testis migration has been completed, but the cauda epididy- Scrotal folds develop early in fetal development. midis and testis remain at some distance from the bottom However, in some species (e.g. bull, horse, and of the developing scrotum. human) they migrate a considerable distance to the www.reproduction-online.org Reproduction (2007) 133 541–561

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final location of the scrotum. This means that the vaginal impinge on target cells and, which actually are process, gubernacular bulb, and epididymis along with obligatory for normal development and function of a testis must follow. The gubernacular bulb is not attached structure. How much of a given molecule actually is to the tissue within the scrotum, until final regression. necessary, versus the amount typically available, is a Directional guidance crucial for inguinoscrotal testis second-level question only indirectly addressed in multi- migration is important in all species. This apparently is dose studies. There are also strain differences in the provided by calcitonin gene-related peptide (CGRP) response of a given tissue to administered hormone released from the genitofemoral nerve (sexually agonists or antagonists. dimorphic, with androgen receptors in cell body) Understanding regulation of testis descent is challen- descending down with the developing gubernaculum ging because of experimental need to provide or and cremaster muscle. Testosterone stimulates pro- eliminate signals at specific points in the process, duction or release of CGRP, which is the chemoattractant almost always, while targeting each of several male and induces the developing tip of the gubernaculum to fetuses in a litter. In most experiments, after admini- grow towards the source of CGRP (Hutson et al. 1998, stration of an agent to a pregnant female, there is a non- Hutson & Hasthorpe 2005, Ng et al. 2005). Assuming uniform response among littermates when evaluated this occurs in all common mammals, factors controlling after birth. Usually this is not commented on or is outgrowth and direction of the genitofemoral nerve attributed to ‘unexplained variation’, and causes are not would have a critical role in final positioning of the testis. sought. Certainly, male members of a litter of rodents Also, lack of testosterone at this time could result in might differ by up to 2 days (11% in late gestation) in malpositioned s.c. testes. stage of development; hence, some might not be within In rodents and rabbits, the striated muscle lining the the targeted window of exposure. It is possible that scrotum is not restricted only to that which had been in microenvironment provided by neighboring male or cremaster muscles of the gubernacular–cremaster female fetuses, and position relative to the cervix, might complex, as the available tissue would be insufficient affect development (Even & vom Saal 1991, Nonneman (Lam et al. 1998). The growing tip of the gubernacular et al. 1992). Regardless of cause, complete reporting bulb in these species includes peripheral myoblasts, would include: tabulation of all phenotypes; gross which lay down muscle just outside the vaginal process appearance of each important structure, not just testes (Elder et al. 1982, Hutson & Hasthorpe 2005). As rodents locations; recognition that litter and not male fetus is and rabbits do not have a narrow (essentially closed) the experimental unit; and statistical consideration inguinal canal after completion of testis descent, the of the non-uniform responses of litters and pups testes can move freely into the abdominal cavity by within litters. Given the nature of published data, we retraction along with inversion of the scrotum. have considered status of the majority of testes as evidence for or against the need for a given enabling molecule or factor. Sequential control of testis descent: mechanisms and what might go wrong Overview of regulatory mechanisms There are a large number of genes and gene products involved in regulation of testis descent (Basrur & Basrur Abdominal translocation of testes is dependent on Insl3 2004, Klonisch et al. 2004). Baumans et al. (1983) to stimulate growth of the gubernaculum to form an hypothesized that a low molecular weight molecule in anchoring structure; testosterone is not required for aqueous extracts of pig testes stimulated gubernaculum completion of this phase. However, testosterone brings development; it was 20 years until Insl3 was identified. about masculinization of the genitofemoral nerve and It is now known, based on data for multiple species, starts to stimulate growth of the vaginal process and that products of Insl3, Great, androgen receptor, and cremaster muscle, to allow completion of the third CGRP genes, and those involved in testosterone phase. production, must be available during critical points Transinguinal migration of testes is dependent on an in development. We will focus on the roles of inguinal canal expanded by the gubernaculum, during these molecules in enabling testis descent, but other the first phase, and movement of an appropriately-sized factors, including intra-abdominal pressure, undoubt- testis through the inguinal canal by intra-abdominal edly are involved. pressure. Neither Insl3 nor testosterone is required for this phase. Inguinoscrotal migration of testes is dependent on proper directional growth of the genitofemoral nerve. Challenges in interpreting data Normally, testosterone enhances secretion of CGRP from When considering regulation of testis descent, it is the genitofemoral nerve to provide direction to guberna- important to distinguish between what signal molecules cular growth, expansion of the vaginal process

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Downloaded from Bioscientifica.com at 10/01/2021 08:57:07PM via free access Understanding cryptorchidism 553 concomitant with limited growth in the inguinal canal testes positioned similarly to those in animals deprived of region to constrict the passageway, growth of the Insl3 stimulation (Rijli et al. 1995); lack of androgen cremaster muscle, and regression of the gubernaculum. probably is not involved because accessory sex glands Testosterone and AMH apparently are not obligatory for and epididymis develop. thinning and elongation of the cranial suspensory ligament, as the abdominal cavity expands, or for expansion of the gubernaculum. The crucial function Role of testosterone of testosterone is to masculinize the genitofemoral nerve Assignment of a proper role to testosterone requires early in embryogenesis, well before completion of careful reading of primary literature, and going beyond abdominal translocation of testes or initiation of the the fact that ‘testis descent was blocked’ in a knockout last two phases of testis descent. animal, by anti-androgen with high affinity for androgen Studies using estrogenic and anti-androgenic receptor (e.g. flutamide), or some other spontaneous or molecules in cattle and pigs, as well as rabbits, rodents induced manipulation. Heynes & Hutson (1995) and humans, establish that there are different time reviewed early literature. In respect to transinguinal windows when Insl3 or testosterone must be available migration of testes, studies with mouse or rat pups to specific developing tissues (references provided exposed to anti-androgen in utero must be interpreted below). During abdominal testis translocation, Insl3 and with knowledge that birth typically occurs before testosterone apparently are provided by Leydig cells to initiation of transinguinal migration of testes in any nearby tissues (possibly including nerve bodies of the male within a litter, and the endpoint in many studies is genitofemoral nerve) via pathways not involving the the anticipated day of birth (e.g. GD 19 for mice, GD 20 general circulation. Initially, both hormones are for rats). In studies where male mice or rats are reared produced under paracrine control or constitutively after natural birth, administration of flutamide usually (Colenbrander et al. 1979, El-Gehani et al. 1998, does not continue (e.g. Shono et al. 1994b, 1996). Pakarinen et al. 2002, Zhang et al. 2004), although in Hence, by PND 2 or 3, flutamide-induced blockage of humans stimulation of fetal Leydig cells by hCG from tissue responses to endogenous androgen probably is maternal blood (Themmen & Huhtaniemi 2000) might minimal. If there is plasticity in when the developmental have a role. In rats there is a surge in intra-testicular signal is received, target tissues might ‘make up’ for concentration of testosterone around GD 19, which previous lack of testosterone stimulation. Recall that, at occurs before LH appearance in fetal blood after GD 20 least in rats, the hypothalamic–pituitary–gonadal axis is (El-Gehani et al. 1998). operational by PND 1 (El-Gehani et al. 1998). We separately examined if testosterone was obligatory for completion of each phase of testis descent. For reports Role of Insl3 where needed data were available, we calculated the Initially, the gubernaculum is short and thin. In mice, percentage of testes completing a given phase of testis Insl3 transcripts are abundant in testes from GD 13.5 descent, as: abdominal translocation – (number of testes through PND 6 (Nef et al. 2000). The gubernaculum below bladder neck, near internal inguinal ring, or further (especially bulb) is rich in the Insl3-receptor Great, but in process)/(2 testes per animal studied); transinguinal other structures involved in testis descent lack the migration – (number of testes below external inguinal receptor. In all species studied, Insl3 is obligatory to ring)/(number of abdominal testes below the bladder bring about gradual expansion of the gubernaculum as it neck or by internal inguinal ring); and inguinoscrotal invades deeper into the abdominal musculature. This migration – (number of testes properly in scrotum)/ expansion is necessary to provide an anchor for normal (number of testes below the external inguinal ring). abdominal translocation of testes. Thereafter, Insl3 Results are in Table 1. Importantly, for several studies probably has no further role in testis descent. Abdominal summarized in Table 1, authors specifically state that testis translocation is blocked by elimination of Insl3 or flutamide affected differentiation of the mesonephric duct Great genes in mice (Klonisch et al. 2004), or adminis- in most animals. There was no report for mice or rabbits tration of estrogenic molecules, to pregnant females, with data allowing calculations. which bind to a-estradiol receptors present in fetal Leydig Abdominal translocation was completed by O89% of cells and suppress transcription of the Insl3 gene (Nef testes in nine trials with rats or pigs (Table 1) despite et al. 2000). In resulting young, testes are positioned in the prenatal or early postnatal exposure to flutamide abdominal cavity well above the internal inguinal ring, (typically injection of pregnant rats with 50 or and gubernacular development is nil. 100 mg/kg per day). In respect to mice, there is evidence Over-expression of the aromatase gene can cause that completion of abdominal translocation of testes is cryptorchidism (Klonisch et al. 2004), probably because delayed on GD 19 (birth), but not blocked, after it raises intra-testicular estradiol. The role of Hoxa10 exposure to flutamide on GD 14–16. Neonatal location gene products in testis descent is uncertain, but male null of the testes in mice exposed to flutamide in utero (refer mice have a long, thin gubernaculum and abdominal Fig. 6 in Shono et al. 1996) is similar to location of testes www.reproduction-online.org Reproduction (2007) 133 541–561

Downloaded from Bioscientifica.com at 10/01/2021 08:57:07PM via free access 554 R P Amann and D N R Veeramachaneni in mice with genetic agenesis of the pituitary gland for abdominal translocation of testes in these species, (Pakarinen et al. 2002), Tfm mice (Hutson 1986, Griffiths even though testosterone might be available to tissues et al. 1993), or androgen-receptor knockout mice containing androgen receptor. Hence, testosterone is not (Yeh et al. 2002, De Gendt et al. 2004, Notini et al. necessary to regress the cranial suspensory ligament so 2005; photos viewed via web). In all these studies, testes that testes can remain near the neck of the bladder. These were found low in the abdominal cavity close to the conclusions are consistent with Hutson et al. (1997). internal inguinal ring, essentially as would be expected Transinguinal migration was completed by O95% of at completion of abdominal translocation. Some papers testes in rat pups or pig fetuses exposed to flutamide noted that development of the gubernaculum and/or (Table 1). Clearly, exposure to flutamide during late vaginal process was subnormal. In two compilations of pregnancy did not block this phase of testis descent. humans with severe androgen insensitivity syndrome, Most of these studies did not directly address the 90% of 32 testes were below the internal inguinal ring question whether testosterone is necessary during (Hutson 1986) and 46% of 176 testes were outside the transinguinal migration of testes. However, data were abdominal cavity (Barthold et al. 2000). Thus, in mice available for rats exposed to flutamide from GD 12 to exposed to flutamide or mice and humans not expressing PND 27 or administered flutamide by injection on PND normal androgen receptor, abdominal translocation is 1–14 (listed under inguinoscrotal migration in Table 1); completed by the majority of testes. all testes completed transinguinal migration and were Assuming that flutamide blocked binding of testo- found in the scrotum at 16–20 week of age. Hence, sterone to androgen receptors in all rat or pig fetuses, transinguinal migration of testes occurs in rat pups, at testosterone-induced mechanisms are not obligatory the normal time or with slight delay, with testosterone-

Table 1 Impact of prenatal or postnatal exposure to flutamide on completion rate through each phase of testis descent.

Time of flutamide exposure species (Referencea) Age examined Completion rate (%) Number testes entering Abdominal translocation of testes Rat 5 GD paradigms (6) PND 28 97 186 Rat GD 10–20 (2) Adult 90 90 Rat GD 12–21 (4) Adult 98 44 Rat GD 16–19 (5) GD 20 100 56 Rat GD 16–19 (5) PND 5 100 46 Rat GD 16–19 (5) PND 30–35 89 46 Rat GD 16–22 (1) PND 30 89 18 Pig GD 65–113 (1) GD 114 100 20 Pig 5 PND paradigms (2) GD 110 95 180 Transinguinal migration of testes Rat 5 GD paradigms (6) PND 28 98 180 Rat GD 10–20 (2) Adult 100 81 Rat GD 12–21 (4) Adult 100 43 Rat GD 16–19 (5) PND 30–35 95 41 Rat GD 16–22 (1) PND 30 100 16 Rat PND 1–14 (2) Adult 100 12 Pig GD 65–113 (1) GD 114 100 20 Pig 5 PND paradigms (3) GD 110 100 171 Inguinoscrotal migration of testes Rat GD 12–16 (6) PND 28 90 30 Rat GD 15.5–17.5 (6) PND 28 53 38 Rat GD 10–20 (2) Adult 54 81 Rat GD 12–21 (4) Adult 58 43 Rat GD 12 – PND 27 (6) PND 28 87 15 Rat GD 16–19 (5) PND 30–35 69 39 Rat GD 16–19 (6) PND 28 83 30 Rat GD 16–22 (1) PND 30 68 16 Rat GD 18–21 (6) PND 28 100 34 Rat GD 21 – PND 7 (6) PND 21 100 26 Rat PND 1–14 (2) Adult 100 12 Rat PND 1–27 (7) PND 28 100 60 Pig GD 35–60 (3) GD 110 78 40 Pig GD 61–74 (3) GD 110 66 35 Pig GD 65–113 (1) GD 114 45 20 Pig GD 75–84 (3) GD 110 30 43 Pig GD 85–100 (3) GD 110 33 30

Flutamide blocks binding of testosterone to androgen receptor. aReferences: (1) Barthold et al. (1996); (2) Kassim et al. (1997); (3) McMahon et al. (1995); (4) Mylchreest et al. (1999); (5) Shono et al. (1994b); (6) Spencer et al. (1991); (7) Spencer et al. (1993).

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Downloaded from Bioscientifica.com at 10/01/2021 08:57:07PM via free access Understanding cryptorchidism 555 binding blocked during gestation or testosterone-binding on GD 75–84 or GD 85–100 (Table 1); only 30–33% of blocked during the postnatal interval when transinguinal testes completed inguinoscrotal migration. This is in migration actually occurs. contrast to a 66–78% completion rate in piglets exposed In pigs, testis descent usually is completed by birth on to flutamide on GD 35–60 or 61–74. Unfortunately, GD 110–114. For pig fetuses exposed in utero to McMahon et al. (1995) did not target GD 95–100 in their flutamide using six paradigms (Table 1), 100% of testes trials. Apparently, testosterone is very important to had passed through the external inguinal ring when enable inguinoscrotal migration of testes in pigs, but as examined on GD 110 or GD 114. Further, fetal in rats must be available to target tissues before initiation decapitation on GD 42 did not affect testis descent in of the event after GD 100; i.e. during GD 80–100. Since 10 of 12 piglets examined on GD 90–113 (Colenbrander exposure of pig fetuses in the GD 65–113 group et al. 1979). Pituitary input was not necessary to provide encompassed the critical GD 80–100 period, blockage sufficient testosterone secretion to enable testis descent. during the latter interval apparently was effective for This is consistent with the conclusion that impairment of 55% of testes (based on 45% completion rate, Table 1). the hypothalamic–pituitary–testis axis (i.e. insufficient Interestingly, on GD 80–100 concentrations of testo- testosterone) does not preclude descent of human testes sterone in serum from fetal pigs apparently is low through the inguinal canal (Hadzˇiselimovic´ et al. 1984). (!0.4 ng/ml; Wensing & Colenbrander 1986). The rat Collectively, except for postnatally castrated rabbits (see and pig data lead to the same conclusion; testosterone is below), cited studies and other data show that testo- not necessary during inguinoscrotal testis migration, but sterone must have minimal if any importance for must be available before initiation of the event. transinguinal migration of testes. Inguinoscrotal testis migration is blocked in null-mice Elder et al. (1982) studied rabbits orchiectomized on lacking GnRH-promoter, GnRH, LH-receptor,orTfm PND 1 and, at examination on PND 14, found that genes and, hence, the drive for testosterone synthesis in striated muscle of the cremaster muscle was poorly Leydig cells is presumed to have minimal constitutive developed and mesenchyme of the gubernaculum had secretory capacity (Hutson et al. 1997, El-Gehani et al. undergone ‘fatty replacement’. Injection of dihydro- 1998, Klonisch et al. 2004). In 80% of cryptorchid boys testosterone prevented this regression and enabled with failure of inguinoscrotal testis migration (testis intussusception of the cremaster muscles, a pivotal step found below the external inguinal ring), the main in transinguinal migration had there been testes. etiological factor was impairment of the hypothalamic– What changes to bring about transinguinal migration pituitary–testis axis (Hadzˇiselimovic´ et al. 1984). In such of testes? We have minimized the role of testosterone as individuals, the problem likely was not caused by lack of a factor facilitating transinguinal migration of a testis testosterone exclusively during inguinoscrotal testis (Table 1), via actions such as stimulating reduction in migration. Rather, the problem likely was caused by size of the gubernaculum, and attributed action of Insl3 lack of testosterone coincident with the initial phase of to the abdominal translocation phase. We assume, as testis descent, when it very rarely impacts abdominal have others (Wensing 1988, Hutson et al. 1997), that translocation of testes, just as in rats and pigs. intra-abdominal pressure exerts sufficient force on a The crucial role of testosterone apparently is mascu- testis, provided it is sufficiently reduced in size, to push linization of the genitofemoral nerve, during the window it against the gubernaculum pre-positioned in, and in fetal development when flutamide was most effective dilating, the inguinal canal. This pressure rapidly (Table 1). Actions of testosterone on other target tissues moves the testis into a s.c. location immediately below cannot be excluded, but any such action(s) is not crucial the external inguinal ring. for completion of testis descent in rats or pigs, and Inguinoscrotal migration was completed by 100% of probably humans. The genitofemoral nerve secretes testes in rats exposed to flutamide just before or during CGRP, which binds to receptors in the growing tip of the time when inguinoscrotal migration actually occurs the gubernacular bulb to stimulate cell proliferation and postnatally (Table 1). However, when flutamide was provide directional guidance for expansion (Hutson & administered during a window spanning GD 15.5–18, in Hasthorpe 2005, Ng et al. 2005). Idiopathic failure or five out of seven studies only 53–69% of testes flutamide-blockage of early masculinization of the completed inguinoscrotal migration (83 and 87% genitofemoral nerve would reduce secretion of CGRP completion in two studies); the most dramatic effect of even if the nerve later was stimulated by testosterone flutamide with rats anywhere in Table 1. This is strong (if available). This is consistent with the observed evidence that inguinoscrotal migration of testes requires separation in time of when flutamide exerts an effect availability of testosterone before, but not during, this (GD 16–18 in rat) on testis descent and when the effect phase of testis descent. manifests (after PND 4 in rat). As discussed previously, In pigs, inguinoscrotal migration of testes starts near similar separation in time was evident in pig data. GD 100–110, and is completed by or shortly after birth Although masculinization of the genitofemoral nerve (Gier & Marion 1970). McMahon et al. (1995) found apparently is the ‘choke point’ for testosterone in testis blockage in most piglets exposed prenatally to flutamide descent, this does not mean that testosterone does not www.reproduction-online.org Reproduction (2007) 133 541–561

Downloaded from Bioscientifica.com at 10/01/2021 08:57:07PM via free access 556 R P Amann and D N R Veeramachaneni stimulate other changes; only that the other changes cells provide tropic factors and extends over an interval usually can be overcome. differing greatly among species. In all species, abdomi- Testosterone also targets the vaginal process to nal translocation is the longest phase of testis descent, stimulate changes needed to constrict/close the inguinal except for dogs in which inguinoscrotal migration occurs canal (Barthold et al. 2000; except in rodents and over an interval of similar length. Generally, there is a rabbits) and the cremaster muscles to stimulate their ‘pause’ during which testes remain at the internal growth (at least in rodents or rabbits). The most important inguinal ring. The time-point when transinguinal testis regressive effect of testosterone is to drive changes in migration is initiated ranges widely among species, but molecular structure of the gubernacular bulb and the in all species transit of testes through the inguinal canal is structure’s virtual elimination leaving a short ligament rapid, requiring !2–4 days. Inguinoscrotal migration of (McMahon et al. 1995, Barthold et al. 2000). In dogs, a testis is a relatively short process in many species, but orchiectomy on PND 1, after full development of the can be prolonged for O2 months in horses or humans. gubernaculum, prevented regression of the gubernacu- Typically, final positioning of testes into the scrotum lum; however, gubernacular regression occurred in occurs before 50% of gestation in cattle; late in gestation castrated pups administered testosterone (Baumans in humans, horses, or pigs; or 3–14 days after birth in et al. 1983). A non-obligatory action to induce final rodents and rabbits. regression of the cranial suspensory ligament cannot be excluded for testosterone. Incidence and nature of cryptorchidism Unfortunately, there is a tendency for researchers and What might cause cryptorchidism? clinicians to be imprecise in recording and reporting From the previous discussion, it is logical to conclude exactly where the testis(es) of a cryptorchid animal was that the primary defect causing failure of testis descent located. Presumably this results from lack of awareness, lies within the testis per se. It failed to produce adequate rather than a sloppy approach to research or clinical amounts of Insl3 and/or testosterone when needed. The medicine. Regardless of the reason, imprecision in data testis controls its own fate, although defects in other often precludes meaningful deductions on possible structures or processes involved in testis descent can causes of the problem in a given male. occur. Aberrant expression of Insl3 and testosterone Published data on prevalence of cryptorchidism are receptors could cause defects with a similar phenotype. summarized in Table 2. Prevalence apparently is !5% One might be tempted to conclude that cryptorchidism in most species and breeds/lines; cryptorchidism might frequently was associated with a detectable change in be more common in pigs. Anecdotal impressions from sequence for one of the ‘important’ genes, or in factors cattle and pig breeders suggest that incidence of regulating their expression in the testis. However, cryptorchidism has not changed substantially over the comprehensive analyses of gene sequences in cryp- past 30 years, but there are no valid estimates of current torchid men revealed that Insl3 or Great genes were prevalence. It is evident (Table 2) that for all species aberrant in only 3–5% of such individuals (Ferlin et al. unilateral cryptorchidism is far more common than 2003, Roh et al. 2003, Klonisch et al. 2004) and aberrant bilateral cryptorchidism, except for one report for dogs androgen receptor or estrogen receptor genes in !16% and a unique situation in deer (next paragraph). of cryptorchid men (Garolla et al. 2005, Yoshida et al. However, the location of undescended testes apparently 2005). Given that most cryptorchid human testes are in a differs among species. For horses, in most reports a s.c. location (Hutson et al. 1992, 1997), defects in the majority of retained testes are stated to be in the pathway for testosterone synthesis should be scrutinized. abdominal cavity. For humans, abdominal retention is Gene sequence studies apparently have not been unusual and most testes are just outside the external undertaken with food-producing or companion animals. inguinal ring or near the neck of the scrotum; i.e. s.c. In humans, perhaps two-thirds of cases self-correct within 3 months, with descent after 3 months unlikely (Hutson et al. 1997, Barthold & Gonzalez 2003). This also is Comparison of timing of testis descent reported with dogs and horses. Comparisons of testis descent among species are Sitka Black-Tailed Deer (SBTD) were placed on/near facilitated by expressing timing of events as a percentage Kodiak Island, Alaska, in 1924–1934, when 25 founder of gestation length for a given species. Commitment of animals were transplanted from several areas in south- indifferent gonads to be testes is a triggering event (e.g. east Alaska. Their range now covers the entire Kodiak programming of testicular cells and secretion of Insl3 Archipelago. Recently, we reported (Veeramachaneni and testosterone). In many species this occurs during the et al. 2006; values below include recent unpublished first third of gestation, but it occurs later in dogs and data) that on the low-lying Aliulik Peninsula (w14! especially rodents and rabbits (Fig. 8). Abdominal 50 km) of Kodiak Island, 109 out of 161 SBTD shot by translocation of the testis is initiated as fetal testicular hunters, and carefully examined, were bilateral

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Figure 8 Approximate timing of events involved in testis descent in common mammals. For each species, consensus gestational age when events were detectable in fetuses or newborn were scaled relative to gestation length. The exactitude of demarcations in this figure mask the variation and uncertainty in when an event starts or is completed due to differences in development among individual fetuses, error in establishing gestational age, the dissection process, investigator interpretation, and how data are presented in publication. Although imperfect, this approach provides far better comparisons across species than use of absolute time for each species. Testis formation and differentiation (Fig. 1) occurs during 2–10 days immediately proximate to the bars for each species, and would be when an endocrine disruptor agent could irrevocably affect a testis. Time required for abdominal translocation of testes ranges from w16% in cattle to w57% horses, and appears to be independent of the relative length of the “pause” (orange) between the abdominal translocation and transinguinal migration phases of testis descent. The relatively long interval shown for transinguinal testis migration for humans and horses probably reflect subject/examiner variation rather than a truly long process in a given individual, although the horse inguinal canal is longer than in other species shown. In rabbits and rodents, changes in the structures during transinguinal migration of testes are slightly different than in other species, and requires intussusception of the cylindrical cremaster muscle around the proper portion of the gubernaculum (Fig. 3). Length of gestation, in days, was assumed to be: cattle, 281; mule deer, 203; human, 268; horse, 337; pig, 114; dog, 60; rabbit, 31; and mouse, 20. Compiled from the literature. Note: Klonisch et al. (2004) present a comparative view of testis descent in their Fig. 6, but relative timing of abdominal testis translocation (and also transinguinal plus inguinoscrotal migration) for some species is quite different from that herein. Presumably, this reflects different interpretations of published descriptions of fetal dissections in respect to our definitions of abdominal translocation, transinguinal migration, and inguinoscrotal migration of testes vs their definitions of transabdominal and inguinoscrotal testis descent, plus deciding when testes arrive in their final position. cryptorchid (BCO) and 12 were unilateral cryptorchid transforming testicular cells, and affecting development (UCO). All retained testes were in the abdominal cavity, of the primordial antler pedicles in some males. They typically part way between the kidney and internal recognized that, alternatively, the phenomenon might be inguinal ring and associated with a thin, underdeveloped a residual epigenetic effect altering/blocking expression gubernaculum. This 75% incidence of cryptorchidism is of Insl3, Great, and/or other genes, consequent to extraordinary as is the 90% predominance of bilateral historic exposure of a founder(s) to an estrogenic EDA. cryptorchidism (Table 2). We are unaware of any other Additional research in both areas is commencing. non-experimental population containing 75% cryp- torchid animals. A high proportion of these animals had testicular neoplasia and abnormal antlers. Elsewhere Elimination of cryptorchidism on the Kodiak Archipelago, 12% of 78 SBTD examined Dogma is that cryptorchidism is a heritable condition, were cryptorchid (three UCO and six BCO). despite the lack of a strong basis for this thought. It is also With evidence then available, Veeramachaneni et al. possible that at least some cases of cryptorchidism in (2006) considered alternative potential causes for the common animals result from fetal exposure to endocrine extraordinary prevalence of cryptorchidism among disruptor agent. Despite limited size of studies, there is SBTD on the Aliulik Peninsula. On theoretical grounds no doubt that brother–sister matings of dogs or pigs over they discounted the plausibility of a classic mutation in a several generations increases incidence of cryp- gene(s) essential for testes descent with marked concen- torchidism (Cox et al. 1978, Mikami & Fredeen 1979, tration via inbreeding. Veeramachaneni et al. (2006) McPhee & Buckley 1984). There also is anecdotal hypothesized that it was most likely that this testis-antler opinion that cryptorchidism is familial in some sire dysgenesis resulted from continuing exposure of preg- lines of horses and pigs. nant females to an estrogenic endocrine disruptor agent, There has been no recent breeding study to establish if thereby blocking transabdominal descent of fetal testes, cryptorchidism is a heritable condition. There probably www.reproduction-online.org Reproduction (2007) 133 541–561

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Table 2 Prevalence of cryptorchidism and nature of dysgenesis (percentages unilateral versus bilateral separate from those for testis location).

Phenotype within cryptorchid males (%)

Intra-inguinal Subcutaneous Species Prevalence (%) Unilateral Bilateral Abdominal testis testis testis Referencesa Cat 1.5 84 16 41 59 (19, 29) Cattle !0.5 90 10 34 66 (4, 23, 25) Sitka BT Deerb !12 38 62 (27) 75 10 90 100 0 0 (1) Dogc 1–11 75 25 41 59 (14, 20, 21, 29) 45 55 92 8 (8) Horsed 2–8 93 7 60 39 (6) 86 14 59 41 (22, 24) 90 10 47 53 (15) 81 19 33 67 (9) Human 2–4 66–89 11–34 8 rare 90 (2, 16) 88 12 7 93 (3) 85 15 (5) 70–77 23–30 9 41–72 30–50 (7, 12) Mouse No information on natural occurrence found Pig 12 59 41 (17) 4–5 (18) Rabbit 2 100 0 33 67 0 (26) Sheep 0.1–0.7 33–45 55–67 (10, 11, 13) 4 62 38 (28) aReferences: (1) Amann et al.(nZ161; unpublished); (2) Barthold & Gonzalez (2003); (3) Beltran-Brown & Villegas-Alvarez (1988); (4) Carroll et al. (1963); (5) Cendron et al. (1993); (6) Consensus of combined citations by Leipold (1986); (7) Cortes et al. (2001); (8) Cox et al. (1978); (9) Cox et al. (1979); (10) Dennis (1975); (11) Ercanbrack & Price (1971); (12) Giannopoulos et al. (2001); (13) Gunn et al. (1942); (14) Hayes et al. (1995); (15) FE Hughes of Peterson & Smith, Ocala, FL; personal communication (2006); (16) Hutson et al. (1997); (17) McPhee & Buckley (1984); (18) Mikami & Fredeen (1979); (19) Millis et al. (1992); (20) Nielen et al. (2001); (21) Pearson & Kelley (1975); (22) Rodgerson & Hanson (1997); (23) Spitzer et al. (1988); (24) Stickle & Fessler (1978); (25) St Jean et al. (1992); (26) Veeramachaneni (nZ159, unpublished); (27) Veeramachaneni et al. (2006); (28) Watt (1978); (29) Yates et al. (2003). bThe first row is compiled across 27 areas in Kodiak Archipelago of Alaska, and the second row is for a unique population on the Aliulik Peninsula (average for 8 areas; possible causes of apparently localized phenomenon are discussed in text). cRelative risk of cryptorchidism differed among 25 breeds of dogs (Pendergrass & Hayes, 1975). dRelative risk of cryptorchidism greater in American Saddle and Quarterhorse breeds than in Arabian, Morgan, Thoroughbred, or Standardbred stallions (Hayes, 1986). Some testes designated by authors as intra- inguinal actually might have been subcutaneous. are two reasons. First, mode of inheritance and dam, would impose an unacceptable economic penalty. penetrance are difficult to establish in planned studies, Breeders of race horses, and other companion animals, and essentially impossible to deduce accurately from simply remove an undescended testis from a valuable retrospective analysis. Sire of a cryptorchid male can be UCO, but do not eliminate either parent, brothers, or assumed to be heterozygous for the genes causing the sisters from breeding stock. This mirrors accepted disease, but many matings would be needed to medical practice for humans. determine if a given dam was homozygous or hetero- zygous for each gene involved. Establishing that an animal is a non-carrier for each gene is even more Acknowledgements difficult. Rehfeld (1971) estimated that O40 male offspring would have to be studied at O6 months of Access to historic publications by John Hunter was graciously age to establish that a dam probably was a non-carrier. provided by Simon Chaplin, Senior Curator, Museums of the Second is economic importance; knowledge of the Royal College of Surgeons of England, London, UK. Partial genetics of cryptorchidism would not alter conventional support provided by NIH Grant 1R21-ES014607-01. The authors have nothing to declare in respect to conflicting management practices. financial interests or relationships with any commercial Most cryptorchid bulls are retained for the food chain, product or entity. after castration if appropriate, but UCO bulls usually are not used for breeding. Cryptorchid boars typically are killed neonatally, at minimal economic loss. They are deemed unsuitable for breeding. In a producer References operation, rearing cryptorchid piglets to market weight might result in a carcass with greatly reduced value (due Backhouse KM 1964 The gubernaculum testis Hunteri: testicular descent and maldescent. Annals of The Royal College of Surgeons of to ‘boar odor’ resulting from 5-androst-16-ene-3-one England 35 15–33. produced by remaining testis tissue). To cull non- Backhouse KM & Butler H 1960 The gubernaculum testis of the pig cryptorchid male or female littermates, much less the (Sus scropha). Journal of Anatomy 94 107–120.

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