Expression of AMH in female fetal intersex gonads in the bovine

Anti Müllerian Hormone, AMH, is believed to be the main agent in the freemartin syndrome. Supposing an active role of freemartin gonads in AMH secretion, in the present study, we aimed at investigating the presence and the localization of AMH producing cells either in fetal or in adult freemartin gonads. Our finding of positive AMH cells in a 26-week-old freemartin fetus indicates an active role of masculinized freemartin gonads in AMH secretion. However, the positive reaction, limited to few cells grouped in 'nests' in proximity to testis cord-like structures, supports a chimeric origin of such cells, migrated from the male co-twin. No adult freemartin, irrespective from the degree of masculinization, showed any AMH positive cell.     

Morphological,histological and histochemical studies of the pituitary glands of ovine freemartins

Freemartins are XX/XY chimaeras that develop as a result of the fusion of the placental circulation of at least one male and one female fetus. The pituitary glands of eight normal ewes at various stages of the oestrous cycle and three rams were compared with those of two male-type and three undifferentiated-type freemartins. The pituitaries were heaviest in the male-type freemartins, and their pattern of gonadotrophs, assessed by differential staining, was more intense than in the normal males. The pituitaries of the undifferentiated-type freemartins weighed less than those of the normal ewes but had more stained gonadotrophs than the normal ewes or rams. In both types of freemartins the pattern of cells resembled that of a castrated male.     

The diagnosis of freemartinism in cattle using sex-specific DNA sequences.

The majority of heifers born co-twins to bulls are infertile freemartins. It is important that the condition be diagnosed at an early age as freemartins have no potential for use as replacement stock. A rapid, robust, reliable technique for freemartin diagnosis is described. Three Y-specific polymerase chain reaction (PCR) primer pairs: BOV97M, BRY.1 and AMX/Y were used to detect male cells in the blood of heifers born co-twins to bulls. PCR -based tests have advantages over currently used methods of freemartin diagnosis.     

Cattle twins and freemartin diagnosis

Some cattle breeding improvement programmes particularly in continental Europe aim to reduce the costs of rearing calves by increasing the number suckled by each cow. This encourages the rearing of twins and an increase in the number of heifers born twin-to-bull. Many of these are offered in the market and bought on the assumption that they are single-born. A large proportion will be freemartins and therefore irremediably sterile. Theories of the aetiology of the freemartin condition are summarised. The anatomical, haematological and chromosomal characteristics are described. Diagnostic methods based on these characteristics are described and compared. The clinical method is recommended as reliable in all but a small proportion of cases. The clinical signs of the freemartin at age three to six weeks are the absence of the external os, and a vagina length of 5 inches (12 cm) or less. The veterinary surgeon is encouraged to make the diagnosis on these clinical signs alone.     

Leydig cell hyperplasia in an ENU-induced mutant mouse with germ cell depletion

Repro22 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing depletion of both male and female germ cells. In the present study, we investigated the male phenotypes of the mutant mouse at the adult stage. The repro22/repro22 homozygous mice showed reduced body weights as well as markedly reduced testis weights. Histological examination of the testes at 4 and 10 months of age showed no germ cells in the seminiferous tubules of the affected testis while a number of Sertoli cells were observed in the tubules. In addition to the germ cell depletion, the testes of the affected mouse contained expanded intertubular spaces that were filled by Leydig cell-like interstitial cells. These interstitial cells were confirmed to be Leydig cells by immunohistochmical staining using anti-3beta-HSD antibody. The estimated number of Leydig cells in the affected testes at 10 months of age increased approximately 2 fold compared with those of normal testes. Furthermore, the plasma testosterone levels of the affected mice at 10 months of age were significantly higher than those of the normal mice. These findings indicated that the repro22/repro22 mouse developed hyperplasia of Leydig cells that was presumably caused by the absence of germ cells in the seminiferous tubules.     

A hermaphrodite dog with bilateral ovotestes and pyometra

Hermaphroditism was identified in a 3-year-old American Cocker spaniel with an enlarged os clitoridis that was shown as reddish finger-like structure protruding from the vulva. The urethral orifice was located cranially to the base of the os clitoridis. The gonads were situated caudal to the kidneys at the cranial tips of the uterine horns, and were composed mainly of seminiferous tubules and interstitial cells and had ovarian follicles in the cortices. The uterus was enlarged and revealed pyometra. Gross and histopathological findings of the dog suggested hermaphroditism with bilateral ovotestes and pyometra.     

XX true hermaphroditism in a dog

XX True hermaphroditism was identified in a 5-month-old German Shorthaired Pointer with a large clitoris. The gonads were situated caudal to the kidneys at the cranial tips of the uterine horns, and were composed mainly of seminiferous tubules and interstitial cells and had ovarian follicles in the cortices. Each gonad had efferent tubules, a pampiniform plexus, fimbriae, and a uterine tube. The uterus was positioned normally in the abdomen and had no gross or histologic abnormalities. Giemsa-banded karyotypes revealed a normal female 78,XX chromosomal complement with no structural abnormalities.     

Indurative lymphocytic mastitis in sheep after experimental infection with maedivisna virus

In order to provide further evidence for the association of an indurative lymphocytic mastitis in sheep with MVV (maedi-visna virus) infection, an experimental study was performed. Fourteen MVV-free pregnant ewes, 2 years of age, were divided into two groups. Eight were intravenously inoculated with MVV (strain ZZV-1050); six ewes served as sham-inoculated controls. Post-mortem examinations were carried out at 8, 16 and 28 months. After 8 months, the 3 infected ewes had indurated udders with extensive lymphoid proliferation around lactiferous ducts and in the acinar tissue. The ducts were often partially obliterated. After 16 months, one of the two infected ewes suffered from indurative lymphocytic mastitis. The other was free of specific udder lesions. After 28 months only one of three infected ewes had mild lymphocytic infiltration in the udder. None of the controls, two in each post-mortem session, had lesions typical of this form of mastitis. The lesions were most severe 8 months after infection. At 16 and 28 months lesions were of a lesser degree or were absent. The lung lesions in the infected ewes 8 months after inoculation were similar to the changes in the udder regarding the lymphoid accumulation, although the proliferation around bronchial tree and blood vessels was less pronounced. After 16 and 28 months all infected ewes had peribronchial and perivascular lymphocytic infiltration though of a lesser degree than after 8 months. From these results it is concluded that in addition to the lung and brain lesions MVV infections may cause a specific indurative lymphocytic mastitis.     

Attempted venereal transmission of Chlamydia psittaci in sheep

Attempts were made to transmit Chlamydia psittaci, the causal agent of enzootic abortion of ewes, in three different ways. Ten ewes were inseminated artificially with freshly collected semen containing 10(5) CELD50 chlamydia. Serological evidence of infection was found two weeks before parturition in nine ewes and the organism was recovered from three of them. By six weeks post partum antibody titres had fallen and were negative in six ewes. Twenty-four hours after service two groups of 10 ewes were infected intravaginally with 10(8) CELD50 and 10(3) CELD50 chlamydia respectively. Positive complement fixation titres were present in the first half of pregnancy in all the ewes in the high dose group but not the low dose group. None of the ewes showed evidence of infection at parturition. Fourteen ewes were served by four rams which had been intravenously infected with 10(8) CELD50 chlamydia four to six days earlier. Following service seroconversion occurred but titres became negative again by late pregnancy. No microbiological evidence of infection was detected in any of the ewes at parturition but complement fixation titres were positive in 12 of 14 ewes sampled six weeks post partum. The 14 ewes were sampled during their pregnancies the next year and none showed any evidence of chlamydial infection. It is concluded that venereal transmission of C psittaci is biologically feasible in sheep, but that under normal systems of flock management in Britain it is unlikely to contribute greatly to the epidemiology of enzootic abortion of ewes.     

Anatomical, cytogenetic and behavioural studies of freemartin ewes

The external phenotype and reproductive behaviour of 21 freemartin sheep and two male pseudohermaphrodite sheep were recorded with the aim of identifying any characteristics which might be used to predict a failure to breed. The size and shape of the vulva and clitoris, the length of the vagina, the size of the teats, the presence or absence of inguinal gonads, and the ultrasonographic characteristics of the inguinal gonads were recorded. A subjective assessment of the masculinity of each animal's body form was also made, and its behavioural responses to a virile ram and to an oestrus ewe were recorded. A number of physical and behavioural abnormalities were detected but the only consistent finding in all 23 animals was a short vagina which varied in length from 3.1 to 7.0 cm, compared with 10 to 14 cm in normal animals.     

Indurative lymphocytic mastitis in sheep after experimental infection with maedivisna virus

Summary

In order to provide further evidence for the association of an indurative lymphocytic mastitis in sheep with MVV (maedi‐visna virus) infection, an experimental study was performed. Fourteen MVV‐free pregnant ewes, 2 years of age, were divided into two groups. Eight were intravenously inoculated with MVV (strain ZZV‐1050); six ewes served as sham‐inoculated controls. Post‐mortem exat. inations were carried out at 8, 16 and 28 months.

After 8 months, the 3 infected ewes had indurated udders with extensive lymphoid proliferation around lactiferous ducts and in the acinar tissue. The ducts were often partially obliterated. After 16 months, one of the two infected ewes suffered from indurative lymphocytic mastitis. The other was free of specific udder lesions. After 28 months only one of three infected ewes had mild lymphocytic infiltration in the udder. None of the controls, two in each post‐mortem session, had lesions typical of this form of mastitis. The lesions were most severe 8 months after infection. At 16 and 28 months lesions were of a lesser degree or were absent.

The lung lesions in the infected ewes 8 months after inoculation were similar to the changes in the udder regarding the lymphoid accumulation, although the proliferation around bronchial tree and blood vessels was less pronounced. After 16 and 28 months all infected ewes had peribronchial and perivascular lymphocytic infiltration though of a lesser degree than after 8 months.

From these resutls it is concluded that in addition to the lung and brain lesions MVV infections may cause a specific indurative lymphocytic mastitis.     

羊驼睾丸细胞凋亡及凋亡相关蛋白的定位

本研究旨在观察羊驼睾丸的出生后发育和精子发生过程中的细胞凋亡及凋亡相关蛋白Bcl2和Caspase3 的定位.取材新生、12月龄和24月龄羊驼的睾丸,用TUNEL法检测睾丸发育和精子发生过程的细胞凋亡,用免疫组织化学技术检测凋亡相关蛋白Bcl2和Caspase3在羊驼出生后发育和精子发生过程中的定位.结果显示在新生羊驼睾丸未检测到TUNEL阳性细胞,Caspase3和Bcl2表达于间质细胞,提示在新生期凋亡蛋白参与间质细胞凋亡的调节,为曲精小管的发育提供空间;12月龄羊驼睾丸TUNEL阳性细胞定位于曲精小管中央部分,Caspase3 和Bcl2定位于间质细胞和曲精小管中央生殖细胞,提示在青春期(12月龄)羊驼睾丸,细胞凋亡和凋亡相关蛋白参与曲精小管管腔形成的调节;24月龄羊驼睾丸TUNEL阳性细胞定位于精原细胞、精母细胞和精子细胞,Caspase3 和Bcl2定位于间质细胞和各个发育阶段的生精细胞,Caspase3阳性细胞在精原细胞最高,向精母细胞和精子细胞逐渐减少,Bcl2在精原细胞弱阳性表达,在血睾屏障以内的曲精小管近腔室部分呈弥散性强阳性表达,提示在性成熟(24月龄)羊驼睾丸精子发生过程中,细胞凋亡主要发生于精原细胞和早期精母细胞,Bcl2可能抑制精母细胞之后生殖细胞的凋亡.结果提示在羊驼睾丸出生后发育和精子发生过程中存在细胞凋亡现象;凋亡蛋白Caspase3和Bcl2参与羊驼睾丸发育和精子发生过程中细胞凋亡的调节.     

A Proposed Role for VEGF Isoforms in Sex-Specific Vasculature Development in the Gonad

Many scientists have expended efforts to determine what regulates development of an indifferent gonad into either a testis or ovary. Expression of Sry and upregulation of Sox9 are factors that initiate formation of the testis-specific pathway to allow for both sex-specific vasculature and seminiferous cord formation. Migration of mesonephric precursors of peritubular myoid cells and endothelial cells into the differentiating testis is a critical step in formation of both of these structures. Furthermore, these events appear to be initiated downstream from Sry expression. Sertoli cell secretion of growth factors acts to attract these mesonephric cells. One hypothesis is that a growth factor specific for these cell linages act in concert to coordinate migration of both peritubular and endothelial cells. A second hypothesis is that several growth factors stimulate migration and differentiation of mesonephric 'stem-like' cells to result in migration and differentiation into several different cell lineages. While the specific mechanism is unclear, several growth factors have been implicated in the initiation of mesonephric cell migration. This review will focus on the proposed mechanisms of a growth factor, Vascular Endothelial Growth Factor, and how different angiogenic and inhibitory isoforms from this single gene may aid in development of testis-specific vascular development.     

Growth and reproductive development in freemartins hormonally treated from 1 to 79 weeks of age

Postnatal growth, steroid hormone profiles and response to steroid hormone treatment were studied in 18 freemartins and one normal female born co-twin with a bull. They were either treated postnatally with testosterone or estrone at one week of age, or left untreated until 50 weeks of age when they received silastic implants calculated to release either 12.9 microgram of estrone or 2.6 microgram of estradiol per day per kg of body weight. Later a dihydrotestosterone-treated group was added. Reproductive development was studied by palpation per rectum and by examination when animals were slaughtered at 79 weeks of age. Treated animals grew slightly faster than untreated animals. Testosterone in untreated freemartins averaged 76 and 87 pg/ml of blood plasma during weeks 1 to 48 and 52 to 56. Corresponding values for those animals with small testosterone implants (weeks 1 to 48) and with larger implants (weeks 52 to 56) were 130 and 272 pg/ml. Estrone and estradiol values appeared to fluctuate between 10 and 50 pg/ml but values are uncertain because they were below the sensitivity of the assay then available. Thus, circulating steroid hormone concentrations were similar to those reported for castrates. Testosterone stimulated clitoral development prenatally and postnatally. None of the treatments influenced vaginal depth, which averaged 4.0, 9.0 and 10.9 cm at 1, 24 and 52 weeks of age. Vaginal depth at birth was not a reliable indicator of freemartinism. Androgen may have inhibited udder development, whereas estrogen appeared to be stimulatory. The reproductive organs of the freemartin were characterized by differences in underdevelopment and the general presence of seminal vesicles. The latter structures, plus clitoral development at birth in 3 animals and postnatal response of the clitoris to testosterone is interpreted to indicate that the presence of androgens is one factor in abnormal development. Otherwise, gross morphology of the reproductive tract was not related to hormone treatment, postnatal gonadal histology, endocrinology or lymphocyte chromosomal karyotypes.     

Experimental infection of pregnant ewes with enteric and abortion-source Chlamydophila abortus

Two groups of pregnant ewes were experimentally infected oronasally in midpregnancy. A faecal and an abortion-source isolate of Chlamydophila abortus were used. They were derived from a healthy ewe from a flock with no history of abortion, and from an aborted foetus in a farm with enzootic abortion. As assessed by modified Ziehl-Neelsen (MZN) staining, egg culture, antigen ELISA, the Clearview test and immunohistochemistry, inoculation resulted in placental and/or foetal infection in all ewes. Histopathology revealed placentitis in two and four ewes inoculated with the enteric or abortion-source isolate, respectively, in addition, these samples were immunohistochemically positive for chlamydial antigen. All six ewes infected with the enteric isolate and five of seven ewes infected with the abortion-source isolate showed evidence for a serological response by an indirect ELISA or CFT. Neither chlamydiae nor lesions were detected in the placentae and lambs of the uninfected control ewes, which remained seronegative. Our results suggest that enteric C. abortus can be associated with placental and foetal lesions in sheep.     

A comprehensive study of disorder of sex development in Staffordshire bull terrier dog

An 8‐month‐old female Staffordshire bull terrier was clinically examined because of external sexual organs abnormality—clitoral hypertrophy. As stated by the owner, the female dog had not been in heat yet. Serum profile of testosterone (3.39 ng/ml), as well as an anti‐M?llerian hormone (24.0 ng/ml), suggested the presence of testicular tissue. On the contrary, the estimated level of 17β‐oestradiol (24.6 pg/ml) was approximately two times higher when compared with the normal anoestrus values (5–10 pg/ml). A midline laparotomy was performed to detect the cranial parts of the genital system. Gonads resembling testicle or ovotestis (left) and hypoplastic testicle (right) was visible. Cranial portion of gonads was attached to structures indicative of bilateral epididymidis. The next tubular structures—oviducts were resected along with adherent parts of a hypoplastic uterus. Histological evaluation confirmed that the examined gonad samples were testicles with modified interstitial testicular tissue. Hypertrophy of interstitial space was predominantly formed by Leydig cells. Examination of a cross‐section through the head of suspected epididymidis confirmed their characteristic structures. In addition, the characteristic configuration of the oviducts was presented. The uterus consisted of three walls, in which the endometrium was hypoplastic with the presence of endometrial glands. No Y chromosome was detected by chromosomal analysis using CFA Y probe and the amplification of SRY‐gene coding region (813 bp) indicated genotype 78, XX; SRY‐negative. Sequencing of SOX9 gene exons 1–3 did not reveal any differences in exon 1 and 3. On the contrary, a few changes were determined in the SOX9 exon 2 sequences: G instead of A at position 103; C instead of reference T at position 115; GCG instead of reference CGC at position 138–140; T instead of reference C at positions 161, 164 and 167.     

Comparison of the Distribution of PGP9.5 and NPY in Cryptorchidism and Testicular Tumors in Dogs

The purpose of this study was to analyze the distribution and expression of peptidergic neurotransmitters protein gene product 9.5 (PGP9.5) and neuropeptide Y (NPY) in cryptorchidism and testicular tumors of dogs,compare them with normal testicular tissues of the same age,and provide reference for clinical diagnosis of malignant transformation in testicular tumors of dogs.HE staing,Masson trichrome staining,Gomori silver staining and toluidine blue staining were used to observe the tissue characteristics of reticular fibers,collagen fibers and mast cells.Immunohistochemical SP method and immunofluorescence combined with IPP were used to analyze the expression and localization of PGP9.5 and NPY in tissues.The results showed that the seminiferous epithelium of normal dog testis was composed of 4-7 layers of spermatogenic cells and Sertoli cells,and the distribution of collagen fibers and reticular fibers in interstitial tissue was sparse.The thickness of collagen fibers in the basement membrane of cryptorchidism seminiferous tubules increased,the nucleus of Sertoli concentrated at the base of seminiferous tubules,and the interstitial reticular fibers increased.The tissue structure of testicular tumor was unclear,collagen fibers and reticular fibers were irregularly distributed,and mast cells increased significantly compared with normal and cryptorchid groups.The immunofluorescence results showed that PGP9.5 was moderately positive in Leydig cells of normal testis,no significant expression in spermatogenic cells,strongly positive in Leydig cells and spermatogenic cells of cryptorchidism,and occasional expression in testicular tumors.NPY was occasionally expressed in normal testicular Leydig cells,but not in spermatogenic cells,strong positive expression in Leydig cells and seminiferous epithelium of cryptorchidism,high density and strong positive expression in interstitial vessels,and no obvious expression in testicular tumors.Immunohistochemical statistics showed that the expression of PGP9.5 and NPY in testicular tumor tissue were extremely significantly lower than that in normal group (P<0.01),while the expression of PGP9.5 and NPY in cryptorchidism group were significantly or extremely significantly increased (P<0.05;P<0.01).Therefore,the expression of PGP9.5 and NPY in cryptorchidism of dogs was increased suggesting that the cryptorchidism of dogs had a tendency to develop into a tumor,and was related to the degree of malignant transformation of tumor.     

犬隐睾及睾丸肿瘤中PGP9.5和NPY的分布比较

试验旨在分析肽能神经递质蛋白基因产物9.5（protein gene product 9.5,PGP9.5）和神经肽Y（neuropeptide Y,NPY）在犬隐睾及睾丸肿瘤中的分布和表达,并与同年龄正常睾丸组织进行比较,为认识犬睾丸肿瘤恶变临床诊断提供参考。应用HE染色、Masson三色染色、Gomori银浸染、甲苯胺蓝染色观察各组织中网状纤维、胶原纤维及肥大细胞等组织特征,采用免疫组织化学SP法及免疫荧光法结合IPP统计分析PGP9.5和NPY在组织中的表达及定位。结果显示,正常犬睾丸生精上皮由4~7层生精细胞及Sertoli细胞构成,间质组织胶原纤维和网状纤维分布稀疏。隐睾生精小管基底膜胶原纤维厚度增加,Sertoli细胞核浓缩位于生精小管基底,间质网状纤维增多。睾丸肿瘤组织结构不清晰,胶原纤维和网状纤维无规则分布,肥大细胞较正常组及隐睾组显著增多。免疫荧光定位表明,PGP9.5在正常睾丸Leydig细胞中呈中等阳性表达,生精细胞中无明显表达;隐睾Leydig细胞及生精细胞中呈强阳性表达;睾丸肿瘤中偶有表达。NPY在正常睾丸Leydig细胞中偶见阳性表达,生精细胞中无表达;隐睾Leydig细胞及生精上皮中无表达,间质小血管管壁呈高密度强阳性表达;睾丸肿瘤组织中无明显表达。免疫组化统计表明,睾丸肿瘤组织中PGP9.5和NPY较正常组极显著降低（P<0.01）,隐睾组PGP9.5和NPY表达显著或极显著增加（P<0.05;P<0.01）。因此,犬隐睾时PGP9.5及NPY的表达增高,提示犬隐睾时已有发展为肿瘤的趋势,且与肿瘤恶变程度相关。     

Zur Entwicklung des Pferdehodens

The aim of the study was to answer the open questions concerning the development of the horse's testis. This study revealed that the seminiferous tubules originate from the sex cords of the coelomic epithelium and Leydig cells from the proximal part of mesonephric nephrones, whereas the rete and the ductuli efferentes derive from intermediate and distal parts of the mesonephric tubules. During the development the Leydig cells undergo an enormous proliferation due to the PMSG secretion in the mare. The proliferation of these cells prevent the deep penetration of the rete into the medulla and is therefore the reason for the reduced extension of the rete and mediastinum testis in the stallion, although 80% of these cells degenerate in the last third of pregnancy. The growth of the seminiferous tubules during sexual maturity reduces the rete to the extremitas capitata of the testis.