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The Agu is the only indigenous pig breed in Japan but its population is very small. In order to estimate the efficacy of testicular xenografting for the conservation of Agu pigs, we investigated whether neonatal testicular fragments would acquire the capacity to produce sperm after they had been cryopreserved and grafted into nude mice. Although on day 180 (day 0 = xenografting), grafts showed a low proportion of seminiferous tubule cross-sections containing sperm (0.1 ± 0.1%, mean ± SEM for four mice), the proportion reached 36.9 ± 16.7% (n = 4 mice) by day 240. When single sperm obtained on day 240 was injected into individual porcine oocytes, 28.2% of the oocytes were found to contain one male and one female pronuclei with the second polar body. Moreover, the blastocyst formation rate after injection of the xenogeneic sperm was 28.4%, whereas that in the absence of sperm injection (attributable to parthenogenesis) was 13.3%. These findings suggest that more than half of the blastocysts resulted from fertilization. Thus, testicular xenografting could assist the conservation of Agu pigs by salvaging germ cells present in neonatal testes even after cryopreservation., (© 2020 Japanese Society of Animal Science.)

Testicular xenografting, combined with cryopreservation can assist conservation of the genetic diversity of indigenous pigs by salvaging germ cells from their neonatal testes. Using Meishan male piglets as an example, we examined whether testicular tissue would acquire the ability to produce sperm after cryopreservation and grafting into nude mice (MS group). For comparison, testicular tissue from neonatal Western crossbreed male piglets was used (WC group). Sixty days after xenografting (day 0 = grafting), MS grafts had already developed seminiferous tubules containing sperm, whereas in the WC grafts, sperm first appeared on day 120. The proportion of tubules containing spermatids and sperm was higher in the MS group than in the WC group between days 90 and 120. Moreover, in vitro-matured porcine oocytes injected with a single sperm obtained from the MS group on day 180 developed to the blastocyst stage. The blastocyst formation rate after injection of the xenogeneic sperm was 14.6%, whereas the ratio in the absence of such injection (attributable to parthenogenesis) was 6.7%. Thus, cryopreserved Meishan testicular tissue acquired spermatogenic activity in host mice 60 days earlier than Western crossbreed tissue. Such xenogeneic sperm are likely capable of generating blastocysts in vitro., (© 2018 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

Grafting of testicular tissue into immunodeficient mice makes it possible to obtain functional sperm from immature donor animals that cannot be used for reproduction. We have developed a porcine model of human haemophilia A (haemophilia-A pigs) by nuclear transfer cloning from foetal fibroblasts after disruption of the X-linked coagulation factor VIII (F8) gene. Despite having a recessive condition, female F8 +/- cloned pigs died of severe bleeding at an early age, as was the case for male F8 -/Y cloned pigs, thus making it impossible to obtain progeny. In this study, therefore, we produced sperm from F8 -/Y cloned pigs by grafting their foetal testicular tissue into nude mice. Two F8 +/- female pigs were generated from oocytes injected with xenogeneic sperm. Unlike the F8 +/- cloned pigs, they remained asymptomatic, and delivered five F8 -/Y and four F8 +/- pigs after being crossed with wild-type boars. The descendant F8 -/Y pigs conserved the haemophilia phenotype. Thus, the present F8 +/- pigs show resolution of the phenotypic abnormality, and will facilitate production of F8 -/Y pigs as founders of a strain of haemophilia-A pigs for the development of new therapeutics for haemophilia A. This strategy will be applicable to other genetically modified pigs.

A major goal of testicular xenografting is to salvage germ cells from immature animals that cannot be used for reproduction and generate their offspring. In this study, we investigated whether porcine fetal testicular tissue would acquire the ability to produce sperm with full developmental competence after they had been cryopreserved and grafted into nude mice. Testicular fragments from fetuses at 35, 55 and 90 days postartificial insemination (dpi) were vitrified and stored in liquid nitrogen. Immediately after warming, testicular fragments at each fetal stage were transplanted under the back skin of castrated nude mice (Crlj:CD1-Foxn1 nu ) (35-, 55- and 90-dpi groups, respectively) (day 0 = grafting). Before grafting, the testicular fragments contained seminiferous cords consisting of only gonocytes and Sertoli cells. Histological analyses of the testicular grafts revealed that the differentiation of seminiferous tubules was largely dependent on the time after grafting, and not on donor age. On day 180 in each group, 10-20% of the total number of tubule/cord cross-sections examined had germ cells that had progressed beyond the spermatogonial stage. Fewer than 5% of tubule cross-sections contained elongated spermatids or sperm. Between days 360 and 420, tubule differentiation advanced further, until more than 45% of the tubule cross-sections contained elongated spermatids or sperm. Sperm were recovered for the first time from a single mouse in the 55-dpi group on day 180, although on days 360-420 sperm were recovered from most mice in all of the groups. Serum concentrations of inhibin and testosterone in host mice in all of the groups were higher than those in castrated mice that had received no testicular grafts. Single sperm collected from mice in each group on day 300 or later were injected into individual in vitro-matured oocytes, and these sperm-injected oocytes were transferred to the oviducts of 2 or 3 estrus-synchronized recipient gilts. None of the recipients in any of the groups produced piglets. The present results clearly indicate that porcine fetal testes during the gestational period acquire endocrine and exocrine functions after being cryopreserved and grafted into nude mice. However, the ability of xenogeneic sperm derived from fetal testis to generate piglets was not confirmed in the present study., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Xenografting of immature testicular tissue combined with cryopreservation can preserve and use genetic information of prepubertal animals. For establishment of this new approach, it is essential to clarify whether offspring derived from sperm grown in host mice harboring cryopreserved xenografts show normal reproductive development. This study examined serum profiles of gonadal hormones during sexual maturation in pigs generated by intracytoplasmic sperm injection using sperm derived from cryopreserved xenografts (CryoXeno pigs; three males and three females). We also assessed the reproductive abilities of the male CryoXeno pigs by mating them with conventionally produced (conventional) pigs, and by examining the in vitro fertilizing ability of their sperm. For female CryoXeno pigs, reproductive ability was evaluated by artificial insemination with semen from a conventional boar. During the growth of male CryoXeno pigs, the serum concentrations of inhibin and testosterone showed similar changes (P > 0.17) to those in conventional pigs (n = 4). Histologic analyses of the testes revealed no differences (P > 0.2) in the growth and differentiation of seminiferous tubules between CryoXeno and conventional pigs. Three conventional sows delivered 13.0 ± 1.0 (mean ± standard error of the mean) live piglets after being mated with the three CryoXeno males. Sperm obtained from all CryoXeno pigs had the ability to penetrate oocytes, and these fertilized oocytes reached the blastocyst stage in vitro. During the growth of female CryoXeno pigs, the serum inhibin profile was similar (P > 0.17) to that observed in conventional pigs (n = 5). The first rise in serum progesterone concentration to more than 2 ng/mL was noted at 32.0 ± 2.3 weeks of age in the CryoXeno pigs and at 32.0 ± 3.3 weeks in the conventional pigs, suggesting that both pigs reached puberty at a similar age. After puberty, female CryoXeno pigs farrowed 8.3 ± 1.7 (mean ± standard error of the mean; n = 3) live piglets after artificial insemination with semen from a conventional boar. In conclusion, these findings demonstrate that both male and female CryoXeno pigs have normal reproductive abilities., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cryopreservation of immature testicular tissues is essential for increasing the possibilities of offspring generation by testicular xenografting for agricultural or medical purposes. However, successful production of offspring from the sperm involved has never been reported previously. In the present study, therefore, using intracytoplasmic sperm injection (ICSI), we examined whether xenogeneic sperm obtained from immature pig testicular tissue after cryopreservation would have the capacity to produce live piglets. Testicular fragments from 9- to 11-day-old piglets were vitrified after 10- or 20-min immersion in vitrification solution containing ethylene glycol (EG), polyvinyl pyrrolidone (PVP) and trehalose as cryoprotectants, and then stored in liquid nitrogen for more than 140 days. Thirty nude mice were assigned to each immersion-time group. Testicular fragments were transplanted under the back skin of castrated mice immediately after warming and removal of the cryoprotectants. Blood and testicular grafts were then recovered from the recipient mice on days 60, 120, 180 and 230-350 (day 0 =  grafting). Histological assessment of the testicular grafts and analyses of inhibin and testosterone production revealed no significant differences between the two immersion-time groups, indicating equal growth activity of the cryopreserved tissues. A single sperm obtained from a mouse in each group on day 230-350 was injected into an in vitro-matured porcine oocyte, and then the ICSI oocytes were transferred to the oviducts of estrus-synchronized recipient gilts. One out of 4 gilts that had received oocytes fertilized using sperm from the 10-min immersion group delivered 2 live piglets, and one of another 4 gilts from the 20-min group delivered 4 live piglets. Thus, we have successfully generated porcine offspring utilizing sperm from immature testicular tissues after cryopreservation and transplantation into nude mice. The present model using pigs will be applicable to many large animals, since pigs are phylogenetically distant from the murine recipients.

In the process of establishment of an inbred Duroc pig strain, males with size asymmetry of the testes were frequently observed. To clarify the possible causes of this asymmetry, we examined the testes and epididymides of 67 males of the F4-F7 generations at 35-100 weeks of age. Testicular weights showed a wide variation (120-610 g). When the weights of the testes were compared bilaterally, 35 of the 67 males showed more than a 10% difference. Histological examination of testes from this asymmetry group revealed a range of seminiferous tubule disruption including disappearance of all germ cells, but not Sertoli cells, in the epithelium. Focal lesions associated with the degenerated tubules were observed. Trends of incident fibrosis or hyalinization of these lesions were seen in aged males of the asymmetry group. Besides this abnormality of spermatogenesis, infiltration of mononuclear inflammatory cells around the tubule was frequently observed in the asymmetry group (32.9%, compared with 1.6% in males showing testis symmetry). In severe cases, the inflammatory cells were concentrated in the intertubular region instead of Leydig cells. Cellular infiltration was also observed around the epididymal duct and blood vessels, but its incidence did not differ between the symmetry and asymmetry groups. Testicular testosterone levels were significantly increased in the asymmetry group, but those of E2 and inhibin did not differ between the two groups. These histopathological features indicate that disruption of spermatogenesis after orchitis/epididymo-orchitis could induce testicular atrophy. Genetic predispositions for this trait may cause prevalent retrograde infections, resulting in orchitis/epididymo-orchitis.

In vitro production of embryos, including in vitro maturation, fertilization of oocytes and their subsequent culture to the embryo stage, has become the most popular method of studying gametogenesis and embryogenesis in pigs. As well as their utility for basic studies, these procedures now enable us to generate viable embryos and offspring as a means of conserving genetic resources and rare animal breeds. Recently, more advanced technologies such as xenografting of gonadal (testicular and ovarian) tissues into immunodeficient experimental animals have been developed. In combination with in vitro embryo production techniques, this approach may provide many benefits. We have been carrying out studies to acquire basic information about the application of this method to porcine species, and to improve the existing techniques. Recently, we obtained oocytes from ovarian tissue xenografted and grown in nude mice that had the capacity to be fertilized and the ability to develop into early-stage embryos. We also obtained spermatozoa from the xenografted testicular tissues and injected them intracytoplasmically into in vitro-matured oocytes to produce piglets. Here we discuss the further possibilities of conservation and utilization of porcine gonadal tissue by xenografting into immunodeficient mice., (2011 The Authors. Animal Science Journal © 2011 Japanese Society of Animal Science.)

The difference between transcriptional and translational activities of genes is one of the remarkable features in spermatogenesis. In this study, we characterized MIF4GD (MIF4G domain containing), a possible translational regulator, to uncover the function during rat spermatogenesis. Western blot analysis revealed the presence of two forms of MIF4GD with molecular sizes of 25 and 16 kDa in various tissues including the testis. An additional 20-kDa form of MIF4GD was also found exclusively in the testis. These three proteins were abundantly present in pachytene spermatocytes and haploid spermatids. Importantly, the 16-kDa MIF4GD was distinguished from the 25- and 20-kDa proteins by presence in fractions of 40S subunits and ribosomes of spermatogenic cells according to analysis of subcellular localizations. These results suggest that the 16-kDa MIF4GD may function as a translational regulator in spermiogenesis.

Development of the male gonads is a complex process with interaction of various cells in the gonads including germ, Sertoli, Leydig, and myoid cells. TF is a mutant rat strain showing male pseudohermaphroditism, with agenesis of Leydig cells and androgen deficiency controlled by an autosomal single recessive gene (mp). The mp locus was mapped on the distal region of rat chromosome 7 by linkage analysis, but the gene responsible for the mp mutation has not been identified. In this study, we performed fine linkage mapping and sequence analysis to determine the causative gene of the mp mutation, and performed an immunohistochemical study using a Leydig cell-specific marker to investigate detailed phenotypes of the mutant rats during the testicular development. As a result, we found a missense mutation of the gene encoding Desert hedgehog (Dhh) in the mutant rat, which could result in loss of function of the DHH signaling pathway. Histochemical examination revealed remarkably reduced number of fetal Leydig cells and lack of typical spindle-shaped adult Leydig cell in the mp/mp rats. These phenotypes resembled those of the Dhh-null mice. Additionally, testosterone levels were significantly lower in the mp/mp fetus, indicating androgen deficiency during embryonic development. These results indicate that the mutation of the Dhh gene may be responsible for the pseudohermaphrodite phenotypes of the mutant rat, and that the Dhh gene is probably essential for the development of Leydig cells.

Xenografting of testicular tissue into immunodeficient mice is known to be a valuable tool for facilitating the development of immature germ cells present in mammalian gonads. Spermatogenesis in xenografts and/or in vitro embryonic development to the blastocyst stage after ICSI of xenogeneic sperm has already been reported in large animals, including pigs; however, development of the embryos to term has not yet been confirmed. Therefore, in pigs, we evaluated the in vivo developmental ability of oocytes injected after ICSI of xenogeneic sperm. Testicular tissues prepared from neonatal piglets, which contain seminiferous cords consisting of only gonocytes/spermatogonia, were transplanted under the back skin of castrated nude mice. Between 133 and 280 days after xenografting, morphologically normal sperm were recovered, and a single spermatozoon was then injected into an in vitro matured porcine oocyte. After ICSI, the oocytes were electrostimulated and transferred into estrus-synchronized recipients. Two out of 23 recipient gilts gave birth to six piglets. Here, we describe for the first time that oocytes fertilized with a sperm from ectopic xenografts have the ability to develop to viable offspring in large mammals.

Clarification of the endocrine status of host mice provides us with basic knowledge with which we can manipulate the growth and function of xenografted testicular tissues. We investigated the hormonal profiles of castrated mice grafted with porcine immature testicular tissues from 30 to 210 or more days after grafting (day 0=castration and grafting). The serum follicle stimulating hormone (FSH) concentrations of the host mice declined (P<0.05) from day 60 compared with those of the castrated, ungrafted mice. The serum inhibin and testosterone levels were higher (P<0.05) than those in the castrated, ungrafted mice from days 30 and 90 days, respectively. The inhibin levels further increased (P<0.05) from day 90, during which time the levels were higher (P<0.05) than those in the intact male mice. In the grafts, formation of lumens occurred in the seminiferous cords on day 90 and spermatozoa appeared in the lumens from day 120. However, spermatogenesis in the grafts did not reach the qualitatively normal levels observed in adult boars. The intensity of the immune reaction to inhibin alpha subunits in the Sertoli cells of the grafts decreased with differentiation of the seminiferous tubules. The present findings indicate that a feedback loop was established between the mouse hypothalamo-pituitary axis and the grafted porcine tissues from 60 days post-grafting. The results also indicate that the serum inhibin levels in the host mice remained high even after the appearance of lumens in the seminiferous tubules of the grafted tissues; this is strikingly different to the situation in normal male animals, in which the serum inhibin levels decline at around the time of tubular differentiation. The lack of efferent ducts in the tubules of the grafted tissues probably caused the accumulation of inhibin to be released into the lumens, resulting in high concentrations of circulating inhibin. These high levels of inhibin may directly affect spermatogenic activity and suppress FSH secretion.

A gene for FK506 binding protein 6 (Fkbp6) expresses during a specific stage of male and female meiosis. Disruption of the gene influences male reproduction, i.e. arrests spermatogenesis, but not female reproduction. Using the mouse model (targeted disruption), the role of the gene in homologous chromosome pairing has been demonstrated in a previous study. For further understanding the function of Fkbp6 in chromosome synapsis, we evaluated chromosome pairings during male meiosis in the as/as rat, a spontaneous null mutation, and compared them with those of the mouse model. Electron microscopy of the pachytene nuclei unveiled several types of abnormal chromosome pairing in the rat model, as shown in the mouse previously. The frequencies of aberrant pairings in the knockout mice and mutant rats were 42 of 67 nuclei (62.7%) and 20 out of 74 nuclei (27.0%), respectively. In order to clarify the mechanism of male specific infertility in Fkbp6 deficiency, the localization of gammaH2AX, a marker protein of XY chromosome inactivation during male meiosis, was examined. Immunostaining of gammaH2AX unveiled normal localization of the molecule to XY chromosomes (XY body) in both models, showing the independency of FKBP6 in sex chromosome inactivation. Besides the XY body, focal localization of gammaH2AX was observed in accordance with the unsynapsed chromosomes in both types of null animal. These results indicate the fundamental role of Fkbp6 in homologous chromosome synapsis during male meiosis. In conclusion, male specific infertility under Fkbp6 deficiency remains unsolved.

We investigated the production of inhibin in boars from the infantile to pubertal periods by: (1) measurement of testicular and circulating levels of inhibin, (2) characterization of inhibin forms and (3) localization of inhibin alpha subunits in the testis. Total inhibin levels in the testis increased until 8 weeks of age but then declined to much lower values at 15 weeks. Testicular inhibin A and inhibin B were high until 8 weeks. Circulating levels of total inhibin and inhibin A were also high until 8 weeks, then declined from 10 weeks; inhibin B was not detected, because of low sensitivity of the inhibin B assay. Analyses of inhibin A and inhibin B levels in the eluted fractions obtained from testes after immunoaffinity chromatography and SDS-PAGE showed the presence of a peak of approximately 45 kDa until 10 weeks of age. As the boars aged, the levels of inhibin A and inhibin B increased in the molecular weight region of 29-31 kDa. The fractions corresponding to 29 and 30 kDa suppressed FSH release from rat pituitary cells, but the 45 kDa fraction had no FSH-suppressing activity. Total amounts of inhibin A isolated from the SDS gels were similar to those of inhibin B until 10 weeks of age, but were three times higher than those of inhibin B between 15 and 25 weeks. Further fractionation by reverse phase high-performance liquid chromatography revealed that the 29-31 kDa immunoreactive material was composed of mature forms of inhibin A and inhibin B, in addition to a 26 kDa alpha monomer. Immunohistochemistry indicated that positive immunostaining for the alpha subunits was observed in Sertoli cells from the infantile to pubertal periods. Elongated spermatids also showed positive signals at age 25 weeks. These results clearly indicated that: (1) the boar testis has the ability to produce inhibin A and inhibin B during the infantile period but inhibin A is the predominant form towards puberty and (2) the molecular weight forms of inhibin and the sites of production of inhibin change with testicular development.

F-spondin/vascular smooth muscle cell growth-promoting factor (VSGP), purified from the follicular fluid of adult bovine ovaries, has been identified as a promoter of neuronal differentiation and vascular smooth muscle growth. The objectives of the present study were (1) to clarify whether F-spondin is also produced in the testis, which is ontogenically equivalent to the ovary, and (2) to examine whether production of this protein changes with testicular growth. To isolate F-spondin from the testis, testicular homogenates obtained from 8-week-old boars were sequentially subjected to heparin-Sepharose chromatography, diethylaminoethyl (DEAE)-Sepharose chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The isolated protein had a molecular mass of approximately 110 kDa and was cross-reactive with anti-F-spondin antibody by Western blotting. The purified protein was further characterized by amino acid sequence analysis of its internal peptide. The sequence obtained was GEQCNIVPDN VD, and a homology search indicated that the purified protein is a homologue of rat, human, and bovine F-spondin. By fractionation of the same amounts of testis tissue obtained from 1-, 8-, 16-, and 40-week-old boars, we analyzed age-related production of F-spondin in the testis. Western blotting of the fractions obtained from RP-HPLC revealed the presence of a band at approximately 110 kDa, corresponding to F-spondin, in the testes obtained from boars between 1 and 16 weeks old, but this band was not detected at 40 weeks. These results clearly indicate that (1) the porcine testis produces F-spondin and that (2) production of this protein is evident in the immature porcine testis, but not the adult testis.

We investigated testicular and circulating levels of dimeric inhibins in Holstein bulls from the infantile to postpubertal periods (5 to 50 weeks of age) and examined the relationship between the profiles of circulating dimeric inhibins and FSH. Concentrations of total inhibin and inhibin B in the testis were highest at 4 to 5 weeks of age but decreased gradually as the bulls aged. Testicular inhibin A levels showed a gradual decline to a nadir at 15 to 26 weeks of age, but by 39 weeks, they were high again. The contents of total inhibin, inhibin A, and inhibin B per testis generally increased with age. Fractionation of testicular homogenates obtained from 15-week-old bulls by a combination of immunoaffinity chromatography and SDS-PAGE confirmed the presence of two major molecular weight forms (32 and 45 kDa) of dimeric inhibins in the testes. Circulating levels of total inhibin and inhibin A showed a significant increase in bulls at around 10 to 14 weeks of age compared to the levels between 5 and 7 weeks of age but decreased thereafter. However, immunoreactivity for inhibin B was not detected in the peripheral circulation, probably because of low sensitivity of the inhibin B assays. The concentrations of plasma FSH were high at 5 weeks of age but declined to lower levels between 11 and 40 weeks, and then increased from 41 weeks onward. There was no significant correlation between the plasma levels of FSH and inhibin A or total inhibin. The results clearly indicate that the bull testis produces inhibin A and B and secretes at least inhibin A into the circulation during postnatal development. However, the profile of circulating FSH in bulls shows no reciprocal relationship with the inhibin A or total inhibin profile during the postnatal period.

The ultimate goal of this study was to establish an in vitro system to produce sperms. To pursue this goal, immature porcine testicular cells were cultured in stereostructural form and cultured testicular cord was investigated morphologically. At 4 weeks of age, the seminiferous tubules of the porcine testes consisted of undifferentiated germ cells (gonocytes and undifferentiated spermatogonia) and immature Sertoli cells. The interstitial tissue was largely occupied by Leydig cells. The testes were enzymatically digested, and the dispersed cells were encapsulated with alginate either immediately or after freeze-thawing. The resulting testicular cell cords were cultured for up to 10 weeks. After 2 weeks of culture, Sertoli cells, which were identified by their inhibin-positive reaction in immunohistochemistry, and Leydig cells, which were identified by their morphological characteristics, were observed in the cords. Neither undifferentiated nor differentiated types of germ cells were detected. The number of cells in the cords progressively decreased during the culture period. In order to discover the fate of the Sertoli cells, the level of inhibin in the spent media was determined. Inhibin in the media was at a detectable level after 2 days of culture. The levels increased and peaked at 2 weeks. When frozen-thawed testicular cells were applied to the culture, the peak level was maintained for over 8 weeks, in contrast to the gradual decrease of inhibin level when fresh cells were cultured. These results indicate that the culture conditions can sustain the survival of Sertoli cells. Further improvement is required for proliferation and differentiation of germ cells.