Endocrinological characterization of an ovarian sex cord–stromal tumor with a Sertoli cell pattern in a Japanese Black cow

A Japanese Black cow was evaluated for prolonged post‐partum anestrus and enlargement of the right ovary. Transrectal ultrasonography revealed that the right ovary was markedly enlarged and had a solid appearance, while the left ovary was small and inactive. The presumptive diagnosis was directed towards granulosa‐theca cell tumour (GTCT) which was supported by markedly elevated plasma anti‐Müllerian hormone (AMH; 332.0 ng/ml), oestradiol (E₂; 103.3 pg/ml) and immunoreactive inhibin (ir‐INH; 2.1 ng/ml) in comparison with the diagnostic cut‐off points for bovine GTCTs. Since the cow had been infertile and had swelling of the udder, slaughter was chosen. Histopathological examination revealed that the tumour was an ovarian sex cord–stromal tumour (SCST) with a Sertoli cell pattern. These findings suggest that plasma AMH, ir‐INH and E₂ could be possible biomarkers for bovine ovarian SCST with a Sertoli cell pattern, whereas this case could not be distinguished from GTCTs based on endocrinological profile.     

Anti-Müllerian hormone profiles as a novel biomarker to diagnose granulosa-theca cell tumors in cattle

This study was carried out to evaluate the blood profile and tissue expression of Anti-Müllerian hormone (AMH) as a biomarker for granulosa-theca cell tumors (GTCTs) in cattle. Five cases with unilateral ovarian GTCTs (GTCT group) were investigated in comparison to other groups of Japanese Black cows, which had either cystic ovarian disease (COD group, n=5), a functional corpus luteum on Days 9 to 11 of the estrous cycle (Day 0=estrus; CL group, n=13) or received superovulation treatment (SOT group, n=13). We used transrectal ultrasonography and measured plasma AMH, estradiol-17β (E(2)), progesterone (P(4)) and testosterone (T) levels. Moreover, GTCT tissues were collected and examined by immunohistochemical staining (IHC) for AMH. In the GTCT group, ultrasound images of GTCTs were variable and not definitive. However, the AMH level in the GTCT group (n=3, 58.1 ± 66.3 ng/ml) was significantly higher than in the COD, CL and SOT groups (0.1 ± 0.1 ng/ml for GTCT vs. COD, P<0.05; 0.2 ± 0.1 and 0.3 ± 0.2 ng/ml, respectively for GTCT vs. CL and SOT, P<0.01). The other hormonal levels in the GTCT group had no significant differences compared with the COD or SOT group. Neoplastic granulosa cells labeled with AMH antibody clearly demonstrated a variety of tissue patterns in all cases by IHC. To the best of our knowledge, this is the first study to investigate the blood profile and IHC of AMH in bovine GTCTs. Our findings indicate that AMH may be a novel biomarker to diagnose GTCTs in cattle.     

Equine nonneoplastic abnormal ovary in a draft mare with high serum anti-Müllerian hormone: a case study

We performed a standing hand-assisted laparoscopic ovariectomy in a draft mare that presented with high serum anti-Müllerian hormone (AMH) level and had an enlarged single cystic ovary. Histopathological examination revealed no tumor cell proliferation in the ovary, but the presence of a large ovarian cyst was confirmed. In the diagnosis of abnormal ovaries in mares, a comprehensive assessment should be performed, including the monitoring of ovarian morphology and biomarkers over time, to determine the disease prognosis and treatment plan. The case of this mare with a nonneoplastic abnormal ovary and increased serum AMH level was rare. We suggest that standing hand-assisted laparoscopic ovariectomy is useful for the removal of large ovaries in draft mares.     

Elevated Serum Anti-Müllerian Hormone in an Arabian Mare with Granulosa Cell Tumor

Anti-Müllerian hormone (AMH), produced by granulosa cells, has been reported to be elevated in mares with granulosa cell tumor. A 13-year-old Arabian mare was referred after 3 years of infertility. Rectal and ultrasonographic examination revealed enlargement and multicystic appearance of the left ovary. The mare had substantially higher concentration of AMH (21.6 ng/mL) compared with normal cyclic mares (n = 5; 0.36 ± 0.02 ng/mL). Testosterone (50 pg/mL) concentration was also elevated, but progesterone concentration (0.4 ng/mL) was lowered. Accordingly, the mare was presumptively diagnosed with granulosa-theca cell tumor and a left ovariectomy implemented using ventral midline approach under general anesthesia. Histopathological examination confirmed the diagnosis of granulosa-theca cell tumor. Serum concentrations of AMH decreased to 2 ng/mL and 0.5 ng/mL 2 and 4 weeks after tumor removal, respectively. In conclusion, the current report presents confirmatory evidence that evaluation of serum AMH could be useful for diagnosis of granulosa cell tumor in mare.     

Spontaneous repair of the atrophic contralateral ovary without ovariectomy in the case of a granulosa theca cell tumor (GTCT) affected mare

A 21 year old thoroughbred mare with granulosa theca cell tumor (GTCT) in the right side and atrophic contralateral ovary was investigated in this study. After arrival at our laboratory on 10th December 1999, the clinical diagnosis of GTCT was examined by rectal palpation and ultrasonographic image of ovaries. Plasma from peripheral blood was collected in the breeding and non-breeding seasons for hormonal analysis. The results showed that the contralateral ovary regained normal activity without any treatment of the GTCT affected ovary and contained follicles showing different sizes 19 months later. However, the affected right ovary, which became smaller after 4 months, was totally inactive without any follicle. The observations clearly demonstrate that without any treatment of the GTCT affected ovary, a mare can return to her normal estrous cycle within a certain period in some GTCT cases.     

Changes of serum anti‐Müllerian hormone in a mare with granulosa cell tumour following surgery and reinitiation of follicular activity

Anti‐Müllerian hormone (AMH) has been reported to be elevated in mares with granulosa cell tumour (GCT). An 8‐year‐old Thoroughbred mare was presented for not being observed in oestrus after the beginning of the breeding season. Rectal palpation and ultrasonography revealed enlargement and cystic appearance of the left ovary while the right ovary was small with an anoestrous‐like appearance. The AMH concentration was 694.9 ng/ml. Presumptively diagnosed with GCT, the mare was subjected to tumour removal. Histopathology confirmed GCT. To evaluate changes of AMH concentration following surgery, blood samples were collected immediately prior to surgery, and on Days 1, 2, 4, 8, 16 and 32 after surgery. Thereafter, blood samples were collected monthly and also at the time the mare was observed in oestrus (148 days after tumour removal). The AMH concentrations decreased over the first 2 months after surgery (from 721.2 ng/ml to 0.1 ng/ml). Subsequently, AMH concentration increased and peaked at the time of oestrus expression (0.7 ng/ml). The mare then became anoestrous, and AMH concentration decreased and reached 0.2 ng/ml, which was not significantly different from the mean concentration of AMH in normal anoestrous mares (n = 5; 0.26 ± 0.07 ng/ml). In conclusion, the present report implies the potential use of AMH for determination of the time of follicular resumption in mares after GCT removal.     

Dimeric inhibin concentrations in mares with granulosa-theca cell tumors

OBJECTIVE: To determine whether concentrations of dimeric inhibin (CaCA) are greater in plasma and tumor fluid from mares with granulosa-theca cell tumors (GTCT), compared with concentrations in plasma and equine follicular fluid (eFF) from control mares. ANIMALS: 6 mares with GTCT and 12 clinically normal mares. PROCEDURE: The alphabetaA immunoradiometric assay used 2 antibodies, one against each subunit of inhibin (alpha and betaA subunits). Tumor tissue, tumor fluid, and a single blood sample were collected at the time of surgical removal of the GTCT. A single blood sample was collected from 7 control mares during various stages of the estrous cycle. Five other control mares were ovariectomized when their ovaries contained growing follicles of 25 to 35 mm in diameter. A blood sample and eFF from the largest follicle were collected at the time of ovariectomy. RESULTS: Mares with GTCT had significantly greater plasma concentrations of betabetaA (mean +/- SEM, 0.86 +/- 0.53 ng of recombinant human-alphabetaA/ml), compared with control mares (0.14+/-0.02 ng/ml). Concentrations of alphabetaA in tumor fluid and eFF were similar. Concentrations of alphabetaA were significantly lower after ovariectomy. CONCLUSIONS AND CLINICAL RELEVANCE: Dimeric inhibin concentration was higher in plasma from mares with GTCT than in plasma from control mares. Increased granulosa cell mass and loss of mechanisms regulating alphabetaA release in mares with GTCT likely accounted for the increase in plasma concentrations. Measurement of alphabetaA concentrations may be useful for identifying mares with GTCT.     

Anti‐Muellerian hormone concentration in bitches with histopathologically diagnosed ovarian tumours and cysts

Increased concentrations of Anti‐Muellerian hormone (AMH) can indicate a granulosa cell tumour as shown in women, mares and cows. To investigate AMH to differentiate canine granulosa cell tumour from other ovarian pathologies, we evaluated the ovaries of 63 bitches. Blood serum samples were collected before surgery for AMH analysis. Ovaries were submitted for histopathological examination. Fourteen bitches showed normal ovaries. These bitches had AMH values between 0.12 and 0.99 ng/ml. In 20 bitches ovarian cysts i.e., follicular cysts (n = 8), corpora lutea cysts (n = 7), subsurface cysts (n = 5) were diagnosed. These dogs had AMH values of 0.11–2.09 ng/ml. Bitches with small luteinized follicular cysts had slightly higher AMH values than those without ovarian alteration. In 29 cases ovarian neoplasms i.e., granulosa cell tumour (n = 9), epithelial tumours (n = 16), dysgerminomas (n = 3) and one sarcoma were identified. Anti‐Muellerian hormone values of bitches with an ovarian neoplasm except granulosa cell tumour ranged from 0.18 to 1.18 ng/ml. The AMH values of bitches with granulosa cell tumour ranged from 1.12 to ≤23 ng/ml and were significantly higher (p < .05) than in all of the other bitches. The cut‐off of 0.99 ng/ml gave a sensitivity of 100% and a specificity of 94.44% to diagnose granulosa cell tumour. In conclusion, markedly elevated AMH concentrations in bitches are indicative for a granulosa cell tumour. However, negative testing does not rule out the existence of small one. Differentiation of GCT from luteinized follicular cysts may especially be difficult.     

Two atypical cases of granulosa cell tumours in Belgian Blue heifers

This paper describes two atypical cases of granulosa cell tumours (GCTs) in Belgian Blue heifers. In the first case, an 18-month-old heifer died after ovariectomy, and a granulosa-theca cell tumour was diagnosed post-mortem. The second heifer (31 months of age) was near term and was successfully treated surgically for a malignant granulosa cell tumour. Unfortunately, no further reproductive data are available, as the heifer was sold for slaughter. This is the first report describing GCTs in Belgian Blue beef cattle.     

Endocrinological changes before and after removal of the granulosa theca cell tumor (GTCT) affected ovary in 6 mares

To clarify the endocrinological characteristics of the mares with granulosa theca cell tumor (GTCT), peripheral plasma samples from the 6 mares affected with GTCT were collected before and after the surgical removal of the affected ovary. Concentrations of testosterone (T), follicle stimulating hormone (FSH), luteinizing hormone (LH), immunoreactive-inhibin (ir-INH), progesterone (P) and estradiol-17beta (E(2)) in the plasma samples were measured by radioimmunoassay. Before removal of GTCT in all cases, the concentrations of T were significantly higher than those of normal mares at the breeding and non-breeding seasons, whereas plasma concentrations of FSH, LH, ir-INH, P and E(2) were lower. After surgical removal of the affected ovary, the circulatory concentrations of T was declined, but the concentrations of other hormones were constantly low as compared with those of normal mares. The present study suggests that 1) the source of higher T may be due to the abnormal follicles in ovary of GTCT, 2) in the case of GTCT the elevated level of T is observed due to the lack of aromatase, and 3) the high level of T is a typical characteristics for GTCT in mares. It is also suggested 4) due to the elevated levels of T the concentrations of gonadotropins may be suppressed.     

A giant parovarian cyst in a dog with a granulosa cell tumor

A 13-year old intact female poodle showed persistent signs of estrus. Ultrasonographically, a large cystic mass containing echogenic fluid with a thin wall was observed, and it was 8 cm in diameter. The cystic mass was located at the mesovarium between the left ovary and uterine horn, and it was identified as a giant parovarian cyst. A right ovarian mass was histologically diagnosed as a granulosa cell tumor. Persistent estrogen hormone secretion by the granulosa cell tumor was thought to cause a rapid increase in the size of the parovarian cyst. This rare cystic mass originated from the genital system and was differentiated from the uterus.     

Ovariectomy as treatment for granulosa cell tumor in a heifer

A granulosa cell tumor in a 15-month-old heifer was associated with abnormal udder development, relaxation of the pelvic ligaments, and nymphomanic behavior. After surgical removal of the tumor, the anatomic changes were reversed, and the nymphomanic behavior subsided. The heifer conceived 3 times thereafter and became a productive dairy cow. In previously reported cases of granulosa cell tumor, ovariectomy was unsuccessful in returning the cow to normal breeding.     

Localization of inhibin alpha-, betaA- and betaB-subunits and aromatase in ovarian follicles with granulosa theca cell tumor (GTCT) in 6 mares

To clarify the morphological and immunohistochemical characteristics in mares with granulosa theca cell tumor (GTCT), the localization of inhibin subunits (alpha, betaA, betaB) and aromatase in the granulosa cell layers and theca layers in the ovarian follicles were determined by immunohistochemical staining. The follicles were obtained from the ovaries of 6 mares with GTCT and 4 normal mares as controls. Immunohistochemically, inhibin alpha-subunit was localized in the granulosa cells of all follicles showing different sizes in all GTCT cases and betaA- subunit was localized in two GTCT cases in all sized follicles. But inhibin betaB- subunit and aromatase were not localized in GTCT cases. On the other hand, inhibin alpha-, betaA-, and betaB-subunits and aromatase were localized in the large and medium sized follicles, but inhibin betaA- and betaB-subunits and aromatase were not stained in the small sized follicles in normal cases. These findings suggest that some mares with GTCT can secrete dimeric inhibin (inhibin A), but all GTCT cases cannot secrete inhibin B. By the results of aromatase staining it is clear that testosterone is not converted into estradiol due to the lack of aromatase in the GTCT follicles.     

Premature lactation in a heifer with a sex cord-stromal tumor

A tumor of the ovary was believed to be responsible for initiating lactation in a nonpregnant heifer. The only abnormal clinical sign was milk secretion from all 4 mammary glands and a large mass in the abdomen. Concentrations of estrogen and progesterone were markedly high. The mass was removed and identified as a sex cord-stromal tumor with a granulosa cell pattern.     

Monophasic Teratoma of the Ovarian Remnant in a Bitch

An exploratory laparotomy on a mixed‐breed bitch of an estimated age of 5 years revealed that she had undergone ovariectomy in the past, but a cystic structure was present in the area of the right ovary and a whitish mass, approximately 3 cm in diameter, in the area of the left ovary. These structures were removed together with an apparently normal uterus. Histological examination of the cyst showed a thin layer of connective tissue, while the left ovarian mass revealed ovarian tissue and highly differentiated nervous tissue, confirmed through immunohistochemistry. A presumptive diagnosis of mature ovarian teratoma was made. Although teratomas generally contain recognizable elements from more than one of the three germ cell layers, they can also be monophasic, when there is only one germ layer component. Ovarian teratomas are rare in the dog and never before have been reported in an ovarian fragment.     

双氢睾酮对小鼠卵泡颗粒细胞增殖与抗苗勒管激素表达的影响

【目的】探讨双氢睾酮(DHT)对小鼠颗粒细胞增殖与抗苗勒管激素(AMH)表达的影响。【方法】给3周龄的昆明小鼠注射孕马血清促性腺激素(PMSG,10 IU/只)以获取颗粒细胞,颗粒细胞传代后48 h HE染色鉴定形态,绘制颗粒细胞的生长曲线,免疫荧光法鉴定促卵泡素受体(FSHR)的表达。当第2代颗粒细胞汇合度达到50%时,先用无血清的DMEM/F12培养基饥饿处理12 h,然后在培养基中添加不同浓度的DHT (0、10^-9、10^-8、10^-7、10^-6、10^-5 mol/L),培养48 h后检测颗粒细胞增殖情况,实时荧光定量PCR法及ELISA法分别测定AMH基因及蛋白的表达。在颗粒细胞培养液中分别添加10^-6 mol/L Flutamide (雄激素受体(AR)特异性抑制剂)、10^-7 mol/L DHT、10^-7 mol/L DHT+10^-6 mol/L Flutamide、10^-5 mol/L DHT、10^-5mol/L DHT+10^-8 mol/L 11-ketodihydrotestosterone (AR特异性激动剂),分别记为F、D7、DF、D5、DK组,以不添加药物为对照组。培养48 h后,检测各组颗粒细胞增殖及AMH蛋白含量。【结果】体外培养的小鼠颗粒细胞呈梭形或铺路石状,生长曲线呈S形,细胞普遍表达FSHR;与0 mol/L DHT组相比,10^-8、10^-7 mol/L DHT组细胞增殖分别显著和极显著增加(P＜0.05;P＜0.01),10^-5 mol/L DHT组细胞增殖显著降低(P＜0.05);10^-7 mol/L DHT组AMH基因的相对表达量和AMH蛋白含量均极显著增加(P＜0.01)。与D7组相比,DF组颗粒细胞增殖和AMH蛋白含量均极显著降低(P＜0.01);与D5组相比,DK组颗粒细胞增殖和AMH蛋白含量均极显著升高(P＜0.01)。【结论】10^-7 mol/L DHT能够显著促进颗粒细胞增殖和AMH表达,而10^-5 mol/L显著降低了颗粒细胞增殖以及AMH表达,且AR介导了DHT调控颗粒细胞增殖和AMH表达。     

Standing hand-assisted laparoscopic ovariectomy in 65 mares

Objective: To review the efficacy and safety of unilateral ovariectomy by use of a standing hand‐assisted laparoscopic approach and evaluate the effect of ovary size on posttransection hemorrhage after application of a linear stapling device. Study Design: Case series. Animals: Horses (n=65) aged 2–20 years. Methods: Medical records of mares that had ovariectomy performed by use of a standing hand‐assisted laparoscopic approach were reviewed. Data retrieved were signalment, ovary removed (left, right), ovarian size, ovarian pathology, intraoperative and postoperative complications, and length of hospitalization. Results: Mean ovarian diameter was 17 cm. Histopathology (52 mares) confirmed 41 granulosa cell tumors (79%), 8 ovarian cysts (15%), 1 teratoma (2%), and 2 ovaries (4%) without abnormalities. Hemorrhage was observed laparoscopically in 16 mares after transection of the mesovarium. Complications encountered during surgery included 1 mare collapsing in the stocks and 1 mare that hemorrhaged excessively from the incision. Postoperative complications included 2 cases of mild colic. Overall complication rate was 6%. Conclusions: All attempts to remove the target ovary were successful. The approach is safe for the mare, as complications encountered were similar to those recently reported for other approaches.     

Overview of Anti-Müllerian hormone (AMH) and association with fertility in female cattle

Anti-Müllerian hormone (AMH) is produced by granulosa cells of early-antral follicles found on the ovary. After production, it enters circulation and can be detected from a blood sample with an ELISA. Multiple works have found that circulating AMH is a reliable marker of the antral follicle population (AFP) of an animal as well as directly correlated to an animal's response to a superovulation protocol. Research has also found high repeatability within an animal's oestrous cycle. Further use of AMH may be valuable as a reproductive management tool, based on previous research linking productive life with circulating AMH in heifers and success to various breeding protocols by AMH concentration. The aim of this review was to summarize previous works describing basic function of AMH as well as explore recent research examining AMH as a reproductive tool and measurement of fertility in dairy animals.     

Ovariectomy by left flank approach in prepubertal Nelore (Bos indicus) heifers

The main objective of this study was to evaluate a recumbent left flank approach to bilateral ovariectomy in prepubertal heifers and to develop an optimal surgical technique for this procedure. Both ovaries were removed from 6 Nelore heifers by left flank approach without any complications, except in 1 heifer, which was believed to have had only 1 ovary based on ultrasound and exploration during surgery, but was later found to have a remaining functional ovary. Ovariectomy via left flank approach in recumbent prepubertal heifers is feasible and technically easy. This procedure does not involve special instrumentation and, despite the invasive approach, it allows optimal visualization of the ovaries and uterus.     

Surgical removal of a granulosa cell tumor from a heifer

A twelve month old, noncycling Holstein-Friesian heifer, was examined because of abnormal udder development. Rectal palpation revealed a tense right uterine horn. A solid mass in the right abdomen was detected by ballottement. A granulosa cell tumor of the right ovary was removed and nine months later the animal conceived.