Baiyun Zhou

Germ Cell Development in Neonatal Mouse Testes in Vitro Requires Müllerian Inhibiting Substance

To study the effect of müllerian inhibiting substance on testicular germ cell development, especially on gonocytes, whole testes (156) from newborn mice were cultured for 1 to 7 days in vitro. The synthetic medium contained either 10% fetal calf serum, which itself contains endogenous müllerian inhibiting substance, or transferrin, insulin and retinoic acid. Human recombinant müllerian inhibiting substance, rabbit antiserum against müllerian inhibiting substance and/or normal rabbit serum was added to some cultures. The cultured testes were fixed in Stieves fixative and stained with hematoxylin and eosin, and the numbers and types of germ cells per tubule were counted under a light microscope. Preliminary studies showed that germ cell development in newborn mouse testes was similar in vitro to that observed in vivo, except for delay in vitro. Normal germ cell maturation from gonocytes to primary spermatocytes occurred in testes cultured with 10% fetal calf serum only (i), 10% fetal calf serum plus müllerian inhibiting substance plus anti-müllerian inhibiting substance antibody (ii), 10% fetal calf serum plus normal rabbit serum (iii) and transferrin, insulin and retinoic acid plus müllerian inhibiting substance (iv). Maturation from gonocytes to A-type spermatogonia was arrested in testes cultured with 10% fetal calf serum plus anti-müllerian inhibiting substance antibody (p < 0.01), transferrin, insulin and retinoic acid alone (p < 0.001) and transferrin, insulin and retinoic acid plus müllerian inhibiting substance plus anti-müllerian inhibiting substance antibody (p < 0.001). The results are consistent with the hypothesis that müllerian inhibiting substance may be involved in postnatal gonocyte development and suggest that it may be useful to treat infertility associated with undescended testes.

Failure of gubernacular development in the persistent müllerian duct syndrome allows herniation of the testes

A 4-month-old phenotypic male with persistent müllerian duct syndrome (PMDS) is described. Serum levels of müllerian inhibiting substance (MIS) were undetectable in an enzyme immunoassay, consistent with a in an MIS gene. Normal germ cells were present in the testis, suggesting that subsequent azoospermia found in other patients with this abnormality is caused by a postnatal defect in germ cell development after 4 months of age. The testes were not attached by normal male gubernacula, but were loosely anchored by a long “round ligament” to the inguinal region. Failure of masculinasation of the gubernaculum in this syndrome suggests that MIS is responsible for early gubernacular development, and hence may have a role in testicular descent.

Apoptosis in tracheoesophageal embryogenesis in rat embryos with or without adriamycin treatment.

PURPOSE The aim of this study was to determine whether apoptosis participates in separation of the foregut into trachea and esophagus and to evaluate the potential role of apoptosis in the development of esophageal atresia and tracheoesophageal fistula (EA + TEF) induced by Adriamycin. METHODS Timed-pregnant rats were injected daily with either saline or Adriamycin (2 mg/kg) intraperitoneally on days 6 to 9 of gestation. Paraffin sections were prepared from 31 experimental and 31 control embryos at days 12 and 13 of gestation. Condensed nuclei were identified on the paraffin sections using the TUNEL method. Apoptosis was quantified by counting the positively stained cell nuclei in transverse sections of embryos. RESULTS In day 12 control embryos the number of apoptotic nuclei in both lateral ridges of the foregut was high (15.67 +/- 1.38) but relatively low (4.17 +/- 0.80) in Adriamycin-treated embryos (P< .0001). In day 13 Adriamycin-treated embryos, the number of apoptotic nuclei in the region of the upper esophageal pouch was extremely high (23.78.5 +/- 2.20) compared with no detectable apoptotic nuclei in the control embryos. CONCLUSIONS Apoptosis is required for normal tracheoesophageal embryogenesis and may be an important mechanism to be involved in the embryological development of esophageal atresia and tracheoesophageal fistula.

Normal testicular descent and the aetiology of cryptorchidism

1 Introduction.- 2 Normal Testicular Descent.- 2.1 Two-Stage Hypothesis.- 2.2 Role of Mullerian Inhibiting Substance/Anti-Mullerian Hormone.- 2.3 Genitofemoral Nerve Hypothesis.- 2.4 Role of Calcitonin Gene-Related Peptide.- 2.5 Hypothesis to Explain Normal Testicular Descent.- 3 Cryptorchidism.- 3.1 Animal Models.- 3.2 Hypotheses of Aetiology.- 3.3 Recognizable Causes of Cryptorchidism.- 3.4 Congenital vs Acquired Cryptorchidism.- 3.5 Effects of Cryptorchidism.- 4 Management of Cryptorchidism.- 4.1 Rationale for Treatment and Its Timing.- 4.2 Surgical Treatment.- 4.3 Hormonal Treatment.- 4.4 Possible New Approaches to Treatment.- 5 Conclusions.- 6 References.- 7 Subject Index.

Investigation of the intra-abdominal oesophagus and hiatus in fetal rats with oesophageal atresia and tracheo-oesophageal fistula

Abstract After surgical management of their oesophageal atresia (OA) and tracheo-oesophageal fistula (TOF), most patients exhibit evidence of gastro-oesophageal reflux (GOR) and many have oesophagitis. However, the aetiology of the GOR is still controversial. This study was undertaken to document whether there are congenital abnormalities in the intra-abdominal oesophagus and the hiatus in the fetal rat with OA and TOF following exposure to adriamycin (ADR). Time-pregnant rats were injected daily with either saline or 2 mg/kg ADR intraperitoneally on gestational days (GD) 6–9. The fetuses (n = 56) from 8 litters were harvested on GD 21 for examination. The length of the oesophagus between the diaphragmatic crura and the gastro-oesophageal junction (GOJ) and the sizes of the stomach and the oesophageal hiatus were measured under a dissecting microscope. The length of the oesophagus between the diaphragmatic crura and the GOJ in the ADR-treated fetuses (0.85 ± 0.37 mm) was significantly shorter than in control fetuses (2.41 ± 0.32 mm) (P < 0.0001). The size of the stomach in ADR-treated fetuses (5.30 ± 1.01 mm) was significantly smaller than in the controls (8.07 ± 0.49 mm) (P < 0.001). Moreover, the size of the oesophageal hiatus in ADR-treated fetuses (1.16 ± 0.43 mm) was markedly larger than in the controls (0.32 ± 0.1 mm) (P < 0.0001). These results showed that the congenital abnormalities in ADR-treated rat fetuses may account for the oesophageal functional disorders seen after surgical correction in patients who have OA and TOF.

Critical timing of bladder embryogenesis in an adriamycin-exposed rat fetal model: a clue to the origin of the bladder.

BACKGROUND/PURPOSE Administration of Adriamycin (ADR) in utero to pregnant rats (vaginal plug, day 0) on gestational days (GD) 6 to 9 resulted in the offspring having a cluster of malformations, including absence of bladder in 100% of cases. This study aimed to determine the critical timing of the embryological window in bladder development in this animal model. METHODS Timed-pregnant rats were divided randomly and injected intraperitoneally with ADR at 2 mg/kg on GD 6 to 9; GD 7 to 10; GD 8 to 11; GD 9 to 12; GD 6,8, and 9 (missing GD 7); and GD 6, 7, and 9 (missing GD 8). The control group received saline. Fetuses were harvested near term on GD 21 and dissected under a dissecting microscope and examined for gross anorectal and urogenital anomalies. RESULTS Administration of ADR on GD 6 to 9 (n = 63); GD 7 to 10 (n = 42); and GD 6, 7, and 9 (n = 35) resulted in 100%, 83%, and 77% bladder agenesis respectively, in contrast with 53% and 26% on GD 8 to 11 (n = 36) and GD 6, 8, and 9 (n = 49), respectively. The control (n = 52) and the GD 9 to 12 (n = 27) groups all had normal bladder development. The proportion of other urogenital and anorectal anomalies mirror that of bladder agenesis. CONCLUSION The results showed GD 7 to be the critical embryological timing in which bladder development can be affected by ADR, possibly by targeting the gene that is expressed in the embryonic bladder during this narrow time interval.

Factors in testis descent and causes of cryptorchidism

Testicular descent appears to be the result of two separate morphological processes under different hormonal control. The first, or transabdominal, phase is likely to be governed by Müllerian inhibiting substance or anti-Müllerian hormone but this remains controversial. The second, or inguinoscrotal, phase may be controlled by a calcitonin gene-related peptide released from the genitofemoral nerve under androgen stimulation.

Histomorphometric study on germ cell differentiation of unilateral cryptorchidism in the immature pig

Early spermatogenesis from gonocytes to A-type spermatogonia is inhibited in boys with cryptorchidism. Histological lesions in the undescended testes (UDT) are presumably secondary to the extrascrotal position. The purpose of this study is to determine the abnormalities in number and maturation of germ cells in pigs with undescended testes and to examine the effect of UDT location on early germ cell differentiation. Testicular biopsies from 4-week-old pigs with natural unilateral cryptorchidism (n = 20) were divided into three groups: (1) intraabdominal testes, (2) superficial inguinal pouch testes, and (3) contralateral descended testes (CDT). The testis weight (P < .01) and the area of circular cross-section of the seminiferous tubules (P < .05) in intraabdominal testes was lower than in CDT. The number of germ cells (per tubular cross-section) (P < .05) and A-type spermatogonia (P < .05) in intraabdominal and inguinal testes was significantly lower than in CDT. In contrast, the number of gonocytes in intraabdominal testes was similar to that in CDT. The number of Sertoli cells was similar in the three groups. These results demonstrate that defective transformation of gonocytes to A-type spermatogonia in pigs is similar to that observed in boys with unilateral cryptorchidism. The different locations of UDT in the pig are correlated with the degree of impaired spermatogenesis.

Management of Cryptorchidism

Treatment of the undescended testis is based on the premise that early intervention will prevent secondary degeneration of the testis. The issue remains controversial, with some studies suggesting that orchidopexy done later in childhood is not related to infertility. Puri and O’Donnell (1988) performed semen analysis on 142 men who had an orchidopexy when they were 7–13 years of age. They used WHO standards for semen analysis and found that the fertility potential was related to the original gonadal position: those testes nearest the scrotum had the best quality semen. There are some problems with this study, however, as less than half the eligible subjects were studied (Tamhne and Williams 1989). In addition, some of the patients probably had an acquired abnormality such as a retractile or ascending testis, in which the long-term follow-up suggests similar abnormalities in semen quality but to a less severe degree (Nistal and Paniagua 1984; Rasmussen et al. 1988). An extensive review of the literature on the effect of treatment has failed to show any significant improvement in fertility with orchidopexies between four and 14 years (Chilvers et al. 1986). Unfortunately, most of the 27 papers examined described surgery in adolescence rather than in infancy. At present it is not possible to find reliable human studies showing a successful outcome for surgery in infancy because it is only in recent years that orchidopexy has been performed in this age group and there is a long lag-time before fertility can be determined (20–30 years) or malignancy occurs (30–40 years).

Normal Testicular Descent

The normal process of testicular descent is complex and multi-staged rather than being a simple, one-stage mechanism. Descent of the testis occurs only in mammals. A review of different species shows evolution of the position of the gonad from the urogenital ridge in the abdominal cavity first at the groin and then outside the abdominal wall and into a definitive scrotum (Williams and Hutson 1991b). Migration to an extra-abdominal site has enabled the testis to function at a temperature below that of the rest of the body, with the scrotal testis of the human adapted to 33°C (Zorgniotti 1991).